The homing receptors L-selectin and α₄β₇ integrin facilitate entry of T cells into the gut-associated organized lymphoid tissues such as the mesenteric lymph nodes and Peyer patches. We studied the impact of inactivation of genes encoding these receptors on the ability of purified donor CD4⁺ T cells to induce acute lethal graft-versus-host disease (GVHD) associated with severe colitis in irradiated major histocompatibility complex (MHC)–mismatched mice. Whereas lack of expression of a single receptor had no significant impact on the severity of colitis and GVHD, the lack of expression of both receptors markedly ameliorated colitis and early deaths observed with wild-type (WT) T cells. The changes in colitis and GVHD were reflected in a marked reduction in the early accumulation of donor T cells in the mesenteric lymph nodes and subsequently in the colon. The purified WT donor CD4⁺ T cells did not accumulate early in the Peyer patches and failed to induce acute injury to the small intestine. In conclusion, the combination of CD62L and β₇ integrin is required to induce acute colitis and facilitate entry of CD4⁺ donor T cells in the mesenteric nodes associated with lethal GVHD in allogeneic hosts.