Constitutive activation of Notch signaling is required for the proliferation of a subgroup of human T-cell acute lymphoblastic leukemias (T-ALL). Previous in vitro studies have demonstrated the therapeutic potential of Notch signaling inhibitors for treating T-ALL. To further examine this possibility, we applied a γ-secretase inhibitor (GSI) to T-ALL xenograft models. Treatment of established subcutaneous tumors with GSI resulted in partial or complete regression of tumors arising from four T-ALL cell lines that were also sensitive to GSI in vitro. To elucidate the mechanism of action, we transduced DND-41 cells with the active form of Notch1 (aN1), which conferred resistance to in vitro GSI treatment. Nevertheless, in vivo treatment with GSI induced a partial but significant regression of subcutaneous tumors that developed from aN1-transduced DND-41 cells, whereas it induced complete regression of tumors that developed from mock-transduced DND-41 cells. These findings indicate that the remarkable efficacy of GSI might be attributable to dual mechanisms, directly via apoptosis of DND-41 cells through the inhibition of cell-autonomous Notch signaling, and indirectly via disturbance of tumor angiogenesis through the inhibition of non-cell-autonomous Notch signaling. (Cancer Sci 2009; 100: 2444–2450)