Adoptive transfer of large numbers of tumor-reactive CD8+ cytotoxic T lymphocytes (CTLs) expanded and differentiated in vitro has shown promising clinical activity against cancer. However, such protocols are complicated by extensive ex vivo manipulations of tumor-reactive cells and have largely focused on CD8+ CTLs, with much less emphasis on the role and contribution of CD4+ T cells. Using a mouse model of advanced melanoma, we found that transfer of small numbers of naive tumor-reactive CD4+ T cells into lymphopenic recipients induces substantial T cell expansion, differentiation, and regression of large established tumors without the need for in vitro manipulation. Surprisingly, CD4+ T cells developed cytotoxic activity, and tumor rejection was dependent on class II-restricted recognition of tumors by tumor-reactive CD4+ T cells. Furthermore, blockade of the coinhibitory receptor CTL-associated antigen 4 (CTLA-4) on the transferred CD4+ T cells resulted in greater expansion of effector T cells, diminished accumulation of tumor-reactive regulatory T cells, and superior antitumor activity capable of inducing regression of spontaneous mouse melanoma. These findings suggest a novel potential therapeutic role for cytotoxic CD4+ T cells and CTLA-4 blockade in cancer immunotherapy, and demonstrate the potential advantages of differentiating tumor-reactive CD4+ cells in vivo over current protocols favoring in vitro expansion and differentiation.