Tumor-specific T cells are crucial for immunologic control of malignant disease. T cells can be induced in vivo by vaccination or adoptively transferred after activation ex vivo. We investigated the requirements for generating T cells with optimal antitumor effector functions in a murine lymphoma model. Using adoptive transfer, we show that in vivo efficacy of T cells cannot be predicted by tumor reactivity in vitro. A restricted T-cell receptor beta chain repertoire of T-cell populations stimulated ex vivo against tumor cells was necessary but not sufficient for tumor protectivity. Tumor elimination furthermore required vaccination of donor mice, hence in vivo priming. The in vivo priming step may allow tumor-specific T cells to accumulate in vitro more rapidly and to survive for longer periods after withdrawal of the antigenic stimulus and adoptive transfer. A possible survival benefit of in vivo induced T cells may be ascribed to the responsiveness to homeostatic cytokines and to unique cytokine milieus encountered in vivo. Most importantly, monoclonal T cells cannot inhibit tumor growth. A prerequisite of tumor rejection was the expression of at least 2 T-cell receptor beta chains by transferred T-cell populations. This finding has implications for designing adoptive transfer strategies for the clinic.