While performing a routine supply run, a Biohazard Containment Unit had been offloaded from the Bentonville, a supply shuttle that quickly departed from Pioneer Station's cargo hold, where a fanatical saboteur gained remote access to the containment unit, unleashing a Xenomorph infestation within. In an effort to neutralize the infestation from spreading, Deputy Administrator Maeko Hayes initiated Cerberus Protocol, a containment procedure executed by a matrix of orbital missile platforms designed to keep anything from entering or exiting Lethe, the planet below, and in effect destroyed three ships in its path; the Bentonville, the Baldrin, and a nearby Colonial Marine frigate, the USS Otago.

Desmond Miles, a bartender, is kidnapped by the company Abstergo Industries for use as a test subject in the "Animus," a device that can simulate genetic memory. Abstergo intends to put Desmond in the device to recall the memories of his ancestor, Altaïr Ibn-La'Ahad, a member of the Assassin Brotherhood in the year 1191, who lived during the Third Crusade in the Holy Land. Initially, Desmond has trouble adjusting to the device, but eventually relives Altaïr's exploits over the next several days. The game then primarily changes to Altaïr's point-of-view, with occasional transitions to Desmond, due to problems with the Animus or onset of the Bleeding Effect.

The Arkham Knight was originally Jason Todd, the second person to take the mantle of Robin after Dick Grayson and before Tim Drake. He was thought to have been killed by the Joker. Jason had always possessed an explosive temper with numerous incidents of him going too far in his actions as Robin, despite his natural talents and aptitude under Batman's guidance, with hints in his behaviour that he might one day kill instead of using restraint.