In mice, graft-versus-host reactions, associated with powerful graft-versus-tumor effects, can be achieved without graft-versus-host disease (GVHD) by delayed administration of donor lymphocyte infusions (DLI) to established mixed chimeras. However, GVHD sometimes occurs after DLI in established mixed chimeric patients. In contrast to mice, in which T cell recovery from the thymus occurs prior to DLI administration, human T cell reconstitution following T cell-depleted hematopoietic cell transplantation is slow, resulting in lymphopenia at the time of DLI. We demonstrate in this study that T cell lymphopenia is an independent risk factor for GVHD following DLI in the absence of known inflammatory stimuli. DLI-induced GVHD was prevented in lymphopenic recipients by prior administration of a small number of nonalloreactive polyclonal T cells, insufficient to prevent lymphopenia-associated expansion of subsequently administered T cells, through a regulatory T cell-independent mechanism. GVHD was not inhibited by T cells with irrelevant specificity. Moreover, administration of antibiotics reduced the severity of GVHD in lymphopenic hosts. Accumulation of DLI-derived effector T cells and host hematopoietic cell elimination were markedly diminished by regulatory T cell-depleted, nonalloreactive T cells. Finally, thymectomized mixed chimeras showed increased GVHD following delayed DLI. Collectively, our data demonstrate that in the absence of known conditioning-induced inflammatory stimuli, T cell lymphopenia is a risk factor for GVHD in mixed chimeras receiving delayed DLI. Our data suggest that the predisposition to GVHD can at least in part be explained by the presence of occult inflammatory stimuli due to the absence of T cells to control microbial infections.