The number 72 played a significant role to the modern-day Assassins. Most notably, it was used as the password to gain access to the Colosseum Vault during the retrieval of the Apple of Eden in October 2012.
As evident in the Scrolls of Romulus, the earliest-known user of the number 72 as the password to the Vault was Marcus Junius Brutus, circa 45 BCE. The next known person to open the Vault was Ezio Auditore da Firenze in 1506, to seal the Apple of Eden he possessed within. Due to Desmond Miles' vision of Ezio in the Sanctuary, it was known that Ezio later returned to the Villa Auditore to leave behind the clues to the password, which would allow future Assassins to access the Vault and obtain the Apple.
Ezio left the clue on the Sanctuary staircase, just behind the bookcase in Mario Auditore's study. It consisted of a tetractys, an etching of an equilateral triangle with nine other equilaterals within it – the same marking found on the Colosseum Vault's door – and the numbers 1419, 1420, and 1421, which were only visible to those who possessed the gift of Eagle Vision.
- "The Tetragrammaton. The 72 names of God. You see? They're all contained within three verses: Exodus 19 through 21. And, get this, you'll like this. If you arrange the four Hebrew letters in God's name within an equilateral triangle, their numerical values add up to the same number: 72."
- ―Shaun explaining the password to the other Assassins.[src]
When the group of Assassins first arrived at Monteriggioni and gained access to the Sanctuary, Desmond noticed and read out the numbers written into the wall. Shaun Hastings posited them to indicate years, though he could not think of their significance to each other. After discovering the Apple's location through the use of the Animus, the Assassins pondered on how to access the Vault under the Colosseum. Rebecca Crane discovered that the door required a password, but a power failure cut short their attempts to discover it.
As the Assassins waited in the dark and contemplated their next move, Desmond remembered the markings and pointed them out. With this, Shaun finally discovered the numbers' significance and explained them to the rest of the team; pointing out the 72 names of God found in the Bible's book of Exodus, contained within chapter 14, verses of 19 to 21. Having obtained the password, the Assassins left the Sanctuary and headed for the Colosseum. As Desmond made his way through the ruins of the landmark and the path of the Lair of Romulus underneath, Lucy Stillman realized that the current date, October 10, was exactly 72 days away from the Abstergo Industries satellite launch on December 21.
Though she wondered aloud if it was more than a coincidence, Desmond dismissed her unease, stating that, "Someone probably wanted to make sure we get the password right." The team eventually arrived at the Temple of Juno, where the door to the Vault was located. After briefly wondering if the door would understand English, Desmond spoke "72" aloud, subsequently opening the Vault and allowing the Assassins to enter. As Desmond activated the Apple in the Vault, the Isu Juno told him that his journey would begin on the 72nd day before the awakening.
- Rodrigo Borgia, Maria Auditore, and Israel Putnam all died at the age of 72.
- Shaun Hastings mentioned that the construction on the Colosseum started in the year 72 CE.
- William Miles mentioned in an email to Shaun that the team in Osaka was scheduled to make contact with headquarters in 72 hours, but went "dark".
- Giovanni Mocenigo was the 72nd Doge of Venice.
- In Leonardo da Vinci's famous sketch, the "Vitruvian Man", the space between the man's lower arm to upper leg had an angle of 72 degrees.
- An arch on a bridge in his well-known painting, the Mona Lisa, appeared to display the number 72.
- The most famous of da Vinci's scientific journals, The Codex Leicester, was a 72-page notebook.
- The Latin Rule, the Creed of the Templars written by Hugues de Payens and Bernard de Clairvaux, had 72 clauses.
- During the conclusion of Assassin's Creed: Brotherhood, Shaun mentioned that a Phrygian cap and a Masonic eye came together in only one place, though he was frozen before he could continue. This one place was actually on the Declaration of the Rights of Man and of the Citizen of France, with 17 articles, which was approved in 1789. Coincidentally, 89 minus 17 equals 72.
- The tetractys, the triangular figure displayed on the door to the Colosseum Vault, also featured in the Divine Science chapters of Assassin's Creed: Project Legacy.
- In Assassin's Creed: Revelations, Ezio could train a total of 72 recruits to the rank of Assassin, not including Rhodes.
- Desmond Miles was born on the 72nd day of the year 1987.
- According to the Assassin's Creed II database, San Gimignano had 72 towers in its heyday.
- In Assassin's Creed III, Haytham Kenway arrived in Colonial America after 72 days of travel.
- In 1308, during the trials surrounding the disbandment of the Knights Templar, 72 of the arrested knights testified before Pope Clement V.
- In Assassin's Creed III, on the barrier sealing off the Temple's inner chamber, the tetractys can be seen.
- Assassin's Creed: Initiates notes that Daniel Cross often spent up to 3 days in the Animus, or 72 hours.