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The number 72 plays a significant role in Assassin's Creed: Brotherhood. It is used as the password to gain access to the Colosseum Vault, where it must be said aloud.
History

As evident in the Scrolls of Romulus, the earliest known user of 72 as the password to the Vault was Marcus Junius Brutus, circa 45 B.C. The next known person to open the Vault was Ezio Auditore da Firenze in 1507 to seal the Apple of Eden within.
It is known, due to Desmond's vision of Ezio in the Sanctuary in 2012, that Ezio later returned to the Villa Auditore at an unknown date to leave behind the clues to the password that would allow assassins of the future to access the Vault and obtain the Apple. Ezio left the clues just behind the bookcase in Mario's study. The clues consisted of an etching of an equilateral triangle with nine other equilateral within it - the same marking found on the door of the Colosseum Vault- and the numbers 1419, 1420 and 1421 only visible to those who possessed the gift of eagle vision.
Modern-Day discovery

when the group of assassins first arrived at Monteriggioni, and gained access to the sanctuary, Desmond notified the rest of the team that the numbers 1419, 1420 and 1421 were visible to him in eagle vision just at the top of the stairs leading to the sanctuary. The assassins then arrived at the conclusion that they were probably dates.
After discovering the location of the Apple through the use of the Animus 2.0, the modern-day Assassins pondered how to access the vault.
Rebecca attempted to analyze the previous Animus session to uncover a means as to how the door to the vault could be opened, however her work was cut short due to a power failure. As the assassins waited in the dark and contemplated their next move, Desmond remembered the markings he had encountered when he had first opened the entrance to the sanctuary. Calling the teams attention to the numbers again, Shaun Hastings then discovered the significance of the numbers 1419, 1420, and 1421 and explained to the rest of the team:
- "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 Hastings explains the password to the Modern Day Assassins.[src]
Having obtained the password, the Assassins made their way to the Colosseum. As Desmond made his way through the ruins of the Colosseum and the path of the Lair of Romulus underneath, Lucy realized that their were 72 days till the Templar satellite launch. Wondering if it was a coincidence, Desmond dismissed the fact stating that Someone probably wanted to make sure we get the password right. He eventually emerged in the Santa Maria Aracoeli with the other Assassins. After a free-running puzzle, Desmond activiated an elevator that took them down into Juno's Temple where the door to the Vault was. In front of the door, Desmond said aloud, "72," thus opening the Vault.
Trivia
- Coincidentally, both Rodrigo Borgia and Maria Auditore da Firenze died at the age of 72.
- Construction on the Colloseum started in the year 72 A.D. The entrance into the Temple of Juno underneath the Santa Maria Aracoeli was hidden under the Colosseum.
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