Graft-versus-host disease (GVHD) induced by donor-derived T cells remains the major limitation of allogeneic bone marrow transplantation (allo-BMT). We previously reported that the pan-Notch inhibitor dominant-negative form of Mastermind-like 1 (DNMAML) markedly decreased the severity and mortality of acute GVHD mediated by CD4(+) T cells in mice. To elucidate the mechanisms of Notch action in GVHD and its role in CD8(+) T cells, we studied the effects of Notch inhibition in alloreactive CD4(+) and CD8(+) T cells using mouse models of allo-BMT. DNMAML blocked GVHD induced by either CD4(+) or CD8(+) T cells. Both CD4(+) and CD8(+) Notch-deprived T cells had preserved expansion in lymphoid organs of recipients, but profoundly decreased IFN-γ production despite normal T-bet and enhanced Eomesodermin expression. Alloreactive DNMAML T cells exhibited decreased Ras/MAPK and NF-κB activity upon ex vivo restimulation through the TCR. In addition, alloreactive T cells primed in the absence of Notch signaling had increased expression of several negative regulators of T cell activation, including Dgka, Cblb, and Pdcd1. DNMAML expression had modest effects on in vivo proliferation but preserved overall alloreactive T cell expansion while enhancing accumulation of pre-existing natural regulatory T cells. Overall, DNMAML T cells acquired a hyporesponsive phenotype that blocked cytokine production but maintained their expansion in irradiated allo-BMT recipients, as well as their in vivo and ex vivo cytotoxic potential. Our results reveal parallel roles for Notch signaling in alloreactive CD4(+) and CD8(+) T cells that differ from past reports of Notch action and highlight the therapeutic potential of Notch inhibition in GVHD.