According to the Marine campaign, the game takes place in the year "2145 — ten years after the Hadley's Hope incident", referring to the events seen in Aliens. However, 2145 is actually 34 years before the events of Aliens, which takes place in 2179.
Although blurry and indistinct, the USS Tyrargo appears to have the name "SULACO" written on its hull.
The M56 Smartgun's tracking mechanisms are infrared in nature. However, in Aliens, the Xenomorphs are shown to be invisible to infrared scanning (similarly, Yautja are unable to detect them with their heat-based vision mode). The Smartgun should therefore be unable to track the creatures, yet in the game the weapon is able to lock on to and track the creatures with ease. (It is conceivable, however, that ten years after the Hadley's Hope incident and extensive Xenomorph research as shown in the game, the tracking mechanisms of the Smartgun have been improved.)
During the Marine campaign, Corporal Harrison encounters Prince in a stasis pod within the Implantation Lab and unintentionally releases him. Later, Harrison sees Prince jump onto a dropship and escape. Before witnessing this event, Harrison appears to leave the pod where he discovered Prince and enter another. However, in the Predator campaign, Prince jumps onto the dropship from the same pod where he is released.
In the Gear Room, the Disc's description reads "This throwing weapon homes in on its target with lethal precision, it's spinning blades cutting our prey in half." The word "it's" should be spelt "its", as it is earlier in the sentence.
In the Predator campaign, when the Predator protagonist passes a window, he stops and the game's Alien protagonist arrives and jumps on the glass before leaving. However, in the Alien campaign, the game's Predator protagonist runs by the glass without stopping and the Alien instead stops at the window, without jumping on it.
The sign outside Hadley's Hope gives the colony's population as 159. However, in Aliens, the colony's population is 158 (given both on the sign outside in the extended Special Edition, and when Ripley tells Burke he is responsible for 157 deaths — 157 + Newt = 158).
Towards the end of issue 1, Davis One states that, in addition to himself, there are six Davis units still operational aboard the Europa following the battles with the Xenomorphs. However, by studying the identifiable head numbers of the Davis units in the next two issues, at least eight different Davis units appear — 02, 03, 04 (also identified in dialogue), 07, 09, 18, 21 and 27.
While Facehugger implantation has been inconsistent with it even happening within ten minutes. Defiance issue 06 has it happen literally in moments, as when Dr. Hollis is attacked by a horde of Facehuggers, she has to wait for Zula to open the blast doors which doesn't take long, when they break through, they come upon Hollis pushing back the parasite from her face, leave a slime/blood at her lip from where its ovipositor was. Its then assumed that they had rescued her in time, both by how fast their reacted as well the fact that Davis didn't mentioning anything (as he was watching through a window), however by the end of issue and confirmed in the prologue of issue 7, Hollis was implanted by the facehugger, making the process even more unrealistic than the life-cycle speed in the Aliens vs Predator movies.
When attempting to view a page that doesn't exist, the message "SPECIAL ORDER 404 PRIORITY ONE INSURE RETURN OF WEBPAGE FOR ANALYSIS. ALL OTHER CONSIDERATIONS SECONDARY. CONTENT EXPENDABLE." is displayed. Although this was intentional as a reference to Special Order 937 in Alien, "INSURE" should be "ENSURE".