Costimulation plays a critical role in T-cell activation and amplification of anti-tumor immunity. Although CD28 engagement triggers an early activation signal, activation-induced 4-1BB molecule on T cells transmits a crucial signal for further expansion and maturation of effector cells. In this report, the authors show that costimulation through CD28 and 4-1BB pathways synergistically enhances the therapeutic efficacy of T cells from tumor-draining lymph nodes. Intravenous adoptive transfer of costimulated T cells into mice bearing disseminated micrometastasis of a poorly immunogenic, major histocompatibility complex class I-negative A9P melanoma results in a 60% cure rate. Autopsy of mice that died after unsuccessful treatment revealed tumor growth in the liver, spleen, and skin with minimal or no evidence of pulmonary disease. In contrast, mice that received no treatment or noncostimulated T cells had massive pulmonary tumors, suggesting that adoptively transferred T cells are less effective against growth of extrapulmonary tumors. These results show that costimulation of tumor-draining lymph node T cells through CD28 and 4-1BB increases their potential for cancer immunotherapy and suggests that improper trafficking of tumor-reactive T cells to extrapulmonary sites must be improved to enhance clinical efficacy.