ABSTRACTDespite all the advantages that cell‐cultured influenza vaccines have over egg‐based influenza vaccines, the inferior productivity of cell‐culture systems is a major drawback that must be addressed. BST‐2 (tetherin) is a host restriction factor which inhibits budding‐out of various enveloped viruses from infected host cells. We developed BST‐2‐deficient MDCK and Vero cell lines to increase influenza virus release in cell culture. BST‐2 gene knock‐out resulted in increased release of viral particles into the culture medium, by at least 2‐fold and up to 50‐fold compared to release from wild‐type counterpart cells depending on cell line and virus type. The effect was not influenza virus/MDCK/Vero‐specific, but was also present in a broad range of host cells and virus families; we observed similar results in murine, human, canine, and monkey cell lines with viruses including MHV‐68 (Herpesviridae), influenza A virus (Orthomyxoviridae), porcine epidemic diarrhea virus (Coronaviridae), and vaccinia virus (Poxviridae). Our results suggest that the elimination of BST‐2 expression in virus‐producing cell lines can enhance the production of viral vaccines. Biotechnol. Bioeng.2017;114: 2289–2297. © 2017 Wiley Periodicals, Inc.