Influenza virus-specific CD8+ T lymphocytes (CTL) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in- or outside CTL epitopes has been shown to affect viral recognition by virus-specific CD8+ T lymphocytes. Here we investigated the effect of naturally occurring variation at residues outside the conserved immuno-dominant and HLA*0201 restricted M158-66 epitope located in the influenza virus M1 protein on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M158-66 epitope or a control epitope and a novel purposely developed, in vitro system to co-culture influenza virus-infected cells with T cells were used. It was found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M158-66-specific CTLs than the M gene segment of avian viruses. These findings are in concordance with differential CTL activation observed previously caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M158-66 epitope in HLA-A*0201 positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity.IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8+ T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling immune viral evasion strategies and identifying signs of immune adaptation is important to define the role of CD8+ T lymphocytes in affording protection more accurately. Improving our insight of the interaction between influenza viruses on the one hand and virus-specific CD8+ T lymphocyte immunity on the other, may help advance our understanding of influenza virus epidemiology, may aid risk assessment of potentially pandemic influenza virus strains and may benefit design of vaccines that induce more broadly protective immunity.