Regulatory T cells (Tregs) maintain transplantation tolerance and suppress graft-versus-host disease (GvHD) in humans. We monitored 17 subjects with acute GvHD to determine whether Treg frequency correlates with acute GvHD. We found the percent of CD4+CD25−CD69+ Tregs decreases when acute GvHD develops and increases after acute GvHD is controlled. We next sequentially studied 50 subjects receiving conventional allotransplants. We show a high frequency and increased numbers of CD4+CD25−CD69+ Tregs are associated with a reduced risk of acute GvHD. We also show that CD4+CD25−CD69+ Treg numbers increase substantially early after allografts and that a low percent of CD4+CD25−CD69+Tregs is associated with an increased risk of acute GvHD. Reconstitution of Tregs early post-transplant is associated with less acute GvHD. These data imply that CD4+CD25−CD69+ Tregs are a novel subset of regulatory T cells that may protect against acute GvHD after allotransplants.