Most influenza vaccines are produced in chicken eggs but recent human influenza strains often do not grow well in this substrate. The PER.C6 cell line is an alternative platform for vaccine production. Here we demonstrate that PER.C6 cells faithfully propagate recent clinical isolates, without selecting for mutations in the HA gene. PER.C6 cells support the rescue of recombinant influenza viruses from cDNA. We used sequence data from a surveillance programme to generate a PR8-based seed virus with the HA and NA of a contemporary circulating H3N2 human strain, A/England/611/07 (E611) that did not itself grow in eggs. We engineered mutations that affected receptor-binding, G186V or L194P, into the E611 HA gene. Whilst the L194P mutation conferred efficient growth in eggs, G186V did not. The L194P mutation was also spontaneously selected during egg propagation of E611/PR8 7:1 recombinant virus. This suggests generation of a single recombinant vaccine seed might satisfy manufacturers that utilize either eggs or cells for vaccine production.