CD8+ T cells play a central role in cancer immunosurveillance, and the efficient induction of CTLs against tumor Ags is required for successful immunotherapy for cancer patients. Notch signaling directly regulates the transcription of effector molecules in CTLs. However, it remains unclear whether Notch signaling in CD8+ T cells is required for antitumor CTL responses and whether modulation of Notch signaling can augment antitumor CTL responses. In this study, we demonstrate that signaling by Notch2 but not Notch1 in CD8+ T cells is required for antitumor CTL responses. Notch2 flox/flox mice crossed with E8I-cre transgenic (N2F/F-E8I) mice, in which the Notch2 gene is absent only in CD8+ T cells, die earlier than control mice after inoculation with OVA-expressing EG7 thymoma cells. In contrast, Notch1 flox/flox mice crossed with E8I-cre transgenic mice inoculated with EG7 cells die comparable to control mice, indicating that Notch2 is crucial for exerting antitumor CTL responses. Injection of anti-Notch2 agonistic Ab or delta-like 1-overexpressing dendritic cells augmented the antitumor response in C57BL/6 mice inoculated with EG7 cells. These findings indicate that Notch2 signaling in CD8+ T cells is required for generating potent antitumor CTLs, thus providing a crucial target for augmenting tumor immune responses.