Experiments described here demonstrate that the differentiation of mouse thymocytes into cytotoxic T lymphocytes (CTL) requires interleukin-4 (IL-4). To reach this conclusion, we took advantage of our discovery that human and mouse IL-2 have very different effects on the development of CTL from a functionally immature subset of thymocytes. The lobster agglutinin 1 (LAg1)-negative subpopulation of thymocytes proliferated when cultured with concanavalin A (Con A) + human or mouse IL-2, but these cells became CTL only when cultured with Con A + human IL-2. Furthermore, Con A + human IL-2, but not mouse IL-2, stimulated IL-4 production by cells within this population. Con A-induced cytotoxicity by mature LAgl-positive thymocytes and normal thymocytes was also accompanied by secretion of IL-4. The anti-IL-4 mAb 11B11 inhibited induction of cytotoxicity by all thymocyte populations tested. Taken together these experiments indicate that stimuli which induce cytotoxicity by mouse thymocytes also induce the secretion of IL-4, which is necessary for the differentiation of thymocyte CTL precursors into CTL.