Vaccination remains the best available prophylaxis to prevent influenza virus infections, yet current inadequacies in influenza virus vaccine manufacturing often lead to vaccine shortages at times when the vaccine is most needed, as it was the case during the last influenza virus pandemic. Novel influenza virus vaccine production systems will be crucial to improve public health and safety. Here we report the optimization of influenza B virus growth in the proprietary EB66? cell line, currently in use for human vaccine production. To this end, we collected, curated and sequenced 71 influenza B viruses selected for high diversity in date of isolation and lineage. This viral collection was tested for ability to enter and replicate within EB66? cells in a single cycle assay and appears to readily infect these cells. When the collection was tested for viral progeny production in a multi-cycle assay, we found a large variation from strain to strain. The strains with the top growth characteristics from the B/Victoria and B/Yamagata lineages were selected for vaccine backbone generation using a reverse genetics system. We then showed that these backbones maintain their desirable growth within EB66? cells when the HA and NA from poorly growing strains were substituted for the parental segments, indicating that the selected backbones are viable options for vaccine production in EB66?. Finally, we show that compounds previously reported to enhance influenza virus growth in cell culture also increase virus production in the EB66? cell line.