Alloreactive T cells play a critical role in eliminating hematopoietic malignant cells but are also the mediators of graft-versus-host disease (GVHD), a major complication that subverts the success of allogeneic hematopoietic stem cell transplantation (HSCT). However, induction of alloreactive T cells does not necessarily lead to GVHD. Here we report the development of a cellular programming approach to render alloreactive T cells incapable of causing severe GVHD in both major histocompatibility complex (MHC)–mismatched and MHC-identical but minor histocompatibility antigen–mismatched mouse models. We established a novel platform that produced δ-like ligand 4–positive dendritic cells (Dll4^hiDCs) from murine bone marrow using Flt3 ligand and Toll-like receptor agonists. Upon allogeneic Dll4^hiDC stimulation, CD4⁺ naïve T cells underwent effector differentiation and produced high levels of interferon γ (IFN-γ) and interleukin-17 in vitro, depending on Dll4 activation of Notch signaling. Following transfer, allogeneic Dll4^hiDC-induced T cells were unable to mediate severe GVHD but preserved antileukemic activity, significantly improving the survival of leukemic mice undergoing allogeneic HSCT. This effect of Dll4^hiDC-induced T cells was associated with their impaired expansion in GVHD target tissues. IFN-γ was important for Dll4^hiDC programming to reduce GVHD toxicities of alloreactive T cells. Absence of T-cell IFN-γ led to improved survival and expansion of Dll4^hiDC-induced CD4⁺ T cells in transplant recipients and caused lethal GVHD. Our findings demonstrate that Dll4^hiDC programming can overcome GVHD toxicity of donor T cells and produce leukemia-reactive T cells for effective immunotherapy.