Amino acid substitutions have been identified in the influenza A virus nucleoprotein that are associated with escape from recognition by virus-specific cytotoxic T lymphocytes (CTLs). One of these is the arginine-to-glycine substitution at position 384 (R384G). This substitution alone, however, is detrimental to viral fitness, which is overcome in part by the functionally compensating co-mutation E375G. Here, the effect on viral fitness of four other co-mutations associated with R384G was investigated by using plasmid-driven rescue of mutant viruses. Whilst none of these alternative co-mutations alone compensated functionally for the detrimental effect of the R384G substitution, the M239V substitution improved viral fitness of viruses containing 375G and 384R. The nucleoprotein displays unexpected flexibility to overcome functional constraints imposed by CTL epitope sequences, allowing influenza viruses to escape from specific CTLs.