Graft-versus-host disease (GVHD) remains a frequent and severe complication of allogeneic stem cell transplantation (SCT). One approach to reducing alloreactivity is to deplete the graft of alloreactive T cells. Global T cell depletion results in poor immune reconstitution with high mortality from viral infections and disease relapse. Therefore, an approach to selectively deplete alloreactive T cells without compromising other responses would be highly beneficial. We undertook studies to identify an inducible activation marker expressed on alloreactive effector T cells following culture with HLA-mismatched allostimulators. Compared to other markers, CD134 was superior because of its negative baseline expression and rapid upregulation after activation. Depletion of CD134(+) cells from responder populations dramatically reduced specific alloreactivity as determined by reduction of helper T cell precursor frequencies below the threshold predicting development of clinical GVHD while retaining responses to third-party alloantigens. CD134-allodepleted populations retained effectors specific for the Wilms' tumor (WT1) leukemia antigen as determined by WT1 specific pentamers, and CMV-specific effectors as determined by CMV-specific pentamers and CMV-specific ELISpot. Thus, use of CD134-allodepleted grafts may improve allogeneic SCT by reducing GVHD without loss of pathogen-specific and leukemia-specific immunity.