It is shown that, in a mixed lymphocyte reaction, the production of cytotoxic T lymphocytes (CL) from cytotoxic T lymphocyte precursors (CLP) requires two signals which are separated time. Using a flow cytometer-cell sorter and a vital, fluorescent DNA stain, Hoechst 3342, CLP specific for the stimulator cells can be separated from other CLP and from stimulator cells 12 h after initiation of mixed lymphocyte cultures. These CLP are in a state of partial activation and can produce CL in the absence of stimulator cells if a second signal in the form of a concanavalin A-induced spleen cell supernate factor is added. Specific CL are also generated when the partially activated CLP are cultured with both nude spleen cells and stimulator cells. In this case it appears that an interaction between the stimulator cells and the nude spleen cells leads to production of the second signal.