Graft-versus-host disease (GVHD) is a lethal complication of allogeneic bone marrow transplantation (BMT). Immunosuppressive agents are currently used to control GVHD but may cause general immune suppression and limit the effectiveness of BMT. Adoptive transfer of regulatory T cells (T(regs)) can prevent GVHD in rodents, suggesting a therapeutic potential of T(regs) for GVHD in humans. However, the clinical application of T(reg)-based therapy is hampered by the low frequency of human T(regs) and the lack of a reliable model to test their therapeutic effects in vivo. Recently, we successfully generated human alloantigen-specific CD8(hi) T(regs) in a large scale from antigenically naïve precursors ex vivo using allogeneic CD40-activated B cells as stimulators. We report a human allogeneic GVHD model established in humanized mice to mimic GVHD after BMT in humans. We demonstrate that ex vivo-induced CD8(hi) T(regs) controlled GVHD in an allospecific manner by reducing alloreactive T cell proliferation as well as decreasing inflammatory cytokine and chemokine secretion within target organs through a CTLA-4-dependent mechanism in humanized mice. These CD8(hi) T(regs) induced long-term tolerance effectively without compromising general immunity and graft-versus-tumor activity. Our results support testing of human CD8(hi) T(regs) in GVHD in clinical trials.