Recently we reported that natural killer (NK) cells are critical accessory cells required for the differentiation of alloantigen-stimulated CD8+ T cells into effector cytotoxic T lymphocytes (CTL) in vitro. In this study we provide evidence that NK cells are also required for the generation of influenza virus-specific CTL. Depletion of NK cells from responder human peripheral blood mononuclear cells or mouse splenocytes abolished the induction of influenza A virus-specific CTL under culture conditions. Treatment of C57BL/6 mice with the NK cell-depleting NK1.1 monoclonal antibody (mAb) before primary or secondary immunization with influenza A virus abrogated the capacity of CTL precursors to differentiate into influenza virus-specific CTL effectors in vivo. These results extend our previous findings and demonstrate that NK cells critically influence the induction of antigen-specific CTL, both in vitro and in vivo.