Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation. Pretransplant conditioning regimes cause release of proinflammatory cytokines that stimulate alloreactive donor T cells to attack recipient tissues. IL-10 has been shown to directly downregulate CD4+ T cells by suppressing IL-2 secretion and a critical role played by regulatory T cells has been demonstrated in animal models. One defining cytokine profile for regulatory T cells is the production of IL-10. Release of specific cytokines (IL-10, IL-4 and IFN-gamma) was detected using ELISPOT technology, following stimulation of donor peripheral blood mononuclear cells by recipient (human leukocyte antigen-matched sibling) alloantigen or by mitogen. Correlation between the frequency of cytokine-releasing cells and the development of acute GVHD was investigated. A high frequency of donor cells producing IL-10 in response to recipient alloantigen stimulation correlated with absence of acute GVHD after bone marrow transplant (BMT), while low frequency was strongly associated with severe GVHD. This study presents strong evidence that estimating the frequency of donor alloreactive cells producing IL-10 in response to recipient antigens will provide valuable information prior to BMT regarding potential transplant outcome.