Abstract Generation of killer T cells in murine mixed lymphocyte culture required that prekiller cells be presented with both a killer assisting factor(s) (KAF) and certain cellular alloantigens (presumably H-2K or H-2D) early in the sensitization process. Late addition of either KAF or alloantigen-bearing cells to pre-killer cell cultures decreased the levels of cytotoxicity seen by the fifth day of incubation. In both cases this decrease reflected a delay in the development of cytotoxicity. Periodic removal of KAF from mixed lymphocyte cultures showed that maximum cytotoxicity was obtained when KAF was present along with the alloantigen for the full 5-day sensitization period. However, a significant cytotoxicity could be detected when contact with KAF, in the presence of alloantigen, was allowed for as few as 48 hr of culture. These observations suggest that both alloantigen and KAF must be experienced together to initiate cell mediated lympholysis (CML) development and that full CML development requires the continuous presence of KAF thereafter.