In graft-versus-leukemia (GVL) responses, the cellular subsets and effector mechanisms responsible for cytotoxicity against leukemic cells in vivo remain poorly characterized. A murine model of syngeneic GVL that features CD4(+) and CD8(+) T-cell responses against the MMB3.19 myeloid leukemia cell line has been previously described. MMB3.19 expresses high levels of functional Fas and tumor necrosis factor (TNF) receptors that do not transduce proapoptotic signals. Through the use of perforin- and Fas ligand (FasL)-deficient mice, it was demonstrated that CD4(+) T cells mediate anti-MMB3.19 effects in vivo primarily through the use of FasL and secondarily through perforin mechanisms. Conversely, CD8(+) T cells induce GVL effects primarily through the use of perforin and minimally through FasL mechanisms. Although the in vivo observations of CD8(+) T cells were reflective of their in vitro cytotoxic T lymphocyte (CTL) activity, for CD4(+) T cells, in vitro responses were dominated by the perforin pathway. In addition, the diminished capacity of T cells from perforin- and FasL-deficient mice to lyse MMB3.19 target cells appeared directly related to their deficient cytotoxic functions rather than to defects in activation because these cells were fully capable of mounting proliferative responses to the tumor cells. These findings demonstrate that GVL responses of T-cell subsets can involve preferential use of different cytotoxic mechanisms. In particular, these findings identify a role for both FasL-employing CD4(+) CTLs and the more novel perforin-utilizing CD4(+) T-cell subset in responses against a myeloid leukemia.