Acute graft-versus-host disease (aGVHD) often precludes successful immunotherapy of hematologic malignancies with allogeneic T cells. Therefore, we investigated the effect of immunomodulatory superagonistic anti-CD28 monoclonal antibodies (CD28-SA) on the capacity of allogeneic T cells to mediate both aGVHD and the protective graft-versus-tumor (GVT) response. In vivo pretreatment of donor C57BL/6 mice or short-term in vitro culture of donor lymph node cells with a CD28-SA efficiently protected BALB/c recipient mice from aGVHD. This protection strongly relied on the presence of CD28-SA-activated CD4+ CD25+ Foxp3+ regulatory T cells in the donor T-cell inoculum. With respect to the GVT response, CD28-SA-prestimulated T cells were still as potent in clearing lymphoma cells as were T cells without CD28-SA preactivation. Taken together, our data suggest that CD28-SA stimulation of bulk leukocyte cultures in vitro markedly increases the therapeutic window for adoptive immunotherapy with allogeneic T cells in vivo.