The induction of human influenza virus-specific memory cytotoxic T lymphocytes (CTL) from CTL precursors (CTLp) was investigated using limiting-dilution cultures and cell lines. Differentiation of maximal numbers of CTLp in limiting-dilution cultures required at least three signals: antigen stimulation, interleukin-2 (IL-2), and a differentiation factor distinct from IL-2. Antigen-specific CTLp proliferated in response to antigen stimulation and recombinant DNA-derived IL-2, but often failed to acquire cytolytic activity unless conditioned medium (CM) from mitogen-stimulated peripheral blood mononuclear cell (PBMC) cultures was added to the cultures. Temporal analysis of the requirement for CM indicated that it was providing a late signal for CTLp differentiation. This analysis was confirmed by developing CTLp cell lines, which were found to proliferate in response to IL-2 and antigen but not to exhibit influenza virus-specific cytotoxicity until CM was added.