Beyond its critical role in T cells, T-bet regulates the functions of APCs including dendritic cells and B cells, as well as NK cells. Given that recipient APCs are essential for priming allogeneic T cells and recipient NK or T cells are able to reject allogeneic donor cells, we evaluated the role of T-bet on the host in acute graft-versus-host disease (GVHD) using murine models of allogeneic bone marrow transplantation. T-bet−/− recipients developed significantly milder GVHD than their wild type counterparts in MHC-mismatched or CD4-depedent minor histocompatibility Ag–mismatched models. Allogeneic donor T cells, in particular, CD4 subset, significantly reduced IFN-γ production, proliferation and migration, and caused less injury in liver and gut of T-bet−/− recipients. We further observed that T-bet on recipient hematopoietic cells was primarily responsible for the donor T cell response and pathogenicity in GVHD. T-bet−/− dendritic cells expressed higher levels of Trail, whereas they produced lower levels of IFN-γ and IL-12/23 p40, as well as chemokine CXCL9, resulting in significantly higher levels of apoptosis, less priming, and infiltration of donor T cells. Meanwhile, NK cells in T-bet−/− hosts partially contribute to the decreased donor T cell proliferation. Furthermore, although T-bet on hematopoietic cells was required for GVHD development, it was largely dispensable for the graft-versus-leukemia effect. Taken together with our previous findings, we propose that T-bet is a potential therapeutic target for the control of GVHD through regulating donor T cells and recipient hematopoietic cells.