The link between microbial flora and the shaping of immune responses is being increasingly appreciated, and recent data have uncovered a role for recipient microbiota in the severity of graft-versus-host disease (GVHD). The impact of donor microbiota on T cell-mediated alloresponses and GVHD is not known, however. Using multiple clinically relevant murine models, we analyzed the effect of donor microbiota on the severity of GVHD induced by T cells from specific pathogen-free and germ-free donors, and found that donor microbiota does not alter the expansion or differentiation of alloreactive T cells or the severity of GVHD.