A high mutation rate in RNA viruses facilitates viral replication and escape from immune system surveillance. We used a reverse genetics approach to probe the mechanism underlying immune escape for influenza virus-specific CD8+ T cells responding to the immunodominant DbNP366 epitope. All engineered mutants with a substitution at a critical residue (position 6, P6M) evaded recognition by wild-type DbNP366-specific CD8+ T cells. The mutants NP-M6I and NP-M6T were associated with a substantial “hole” in the naïve TCR repertoire against these endogenous epitopes, and thus minimal primary responses. We solved the structures of the NP-M6I, NP-M6T and compare them with the NP-wt as well as the NP-M6W peptides. The structures revealed that the “hole” in the repertoire against those mutants could be due to altered the conformation of a key residue (His155) in the MHCI binding cleft, offering us a molecular mechanism for viral escape.