Egg-based seasonal influenza vaccines are the major preventive countermeasure against influenza virus. However, their effectiveness can be compromised when antigenic changes arise from egg-adaptive mutations on influenza hemagglutinin (HA). The L194P mutation is commonly observed in egg-based H3N2 vaccine seed strains and significantly alters HA antigenicity. An approach to prevent L194P would therefore be beneficial. We show that emergence of L194P during egg passaging can be impeded by preexistence of a G186V mutation, revealing strong incompatibility between these mutations. X-ray structures illustrate that individual G186V and L194P mutations have opposing effects on the HA receptor-binding site (RBS), and when both G186V and L194P are present, the RBS is severely disrupted. Importantly, wild-type HA antigenicity is maintained with G186V, but not L194P. Our results demonstrate that these epistatic interactions can be used to prevent the emergence of mutations that adversely alter antigenicity during egg adaptation