Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. We recently showed in murine studies and in vitro human models that adoptively transferred invariant natural killer T (iNKT) cells protect from GvHD and promote graft-versus-leukemia effects. The cellular mechanisms underlying GvHD prevention by iNKT cells in humans, however, remain unknown. To study relevant cellular interactions, dendritic cells (DCs) were either generated from monocytes or isolated directly from blood of healthy donors or GvHD patients and co-cultured in a mixed lymphocyte reaction (MLR) with T cells obtained from healthy donors or transplantation bags. Addition of culture-expanded iNKT cells to the MLR induced DC apoptosis in a cell contact-dependent manner, thereby preventing T-cell activation and proliferation. Annexin V/PI staining and image stream assays showed that CD4+CD8-, CD4-CD8+ and double negative iNKT cells are similarly able to induce DC apoptosis. Further MLR assays revealed that conventional DCs (cDCs) but not plasmacytoid DCs (pDCs) could induce alloreactive T-cell activation and proliferation. Interestingly, cDCs were also more susceptible to apoptosis induced by iNKT cells, which correlates with their higher CD1d expression, leading to a bias in favor of pDCs. Remarkably, these results could also be observed in GvHD patients. We propose a new mechanism how ex vivo expanded human iNKT cells prevent alloreactivity of T cells. iNKT cells modulate T-cell responses by selective apoptosis of DC subsets, resulting in suppression of T-cell activation and proliferation while enabling beneficial immune responses through pDCs.