Graft-versus-host disease (GVHD) is a common complication after allogeneic hematopoietic stem cell transplantation. Much of our knowledge regarding GVHD comes from experiments on the mouse hematopoietic system due to ethical and technical constraints. Thus, in vivo GVHD models of the human immune system are required. In this study, we report an effective and reliable protocol for xenogeneic GVHD (xeno-GVHD) model induction using NOD/SCID mice, in which mice underwent a conditioning regimen consisting of intraperitoneal injection of cyclophosphamide and anti-CD122, followed by transfusion of phytohemagglutinin-activated human peripheral blood mononuclear cells containing 1 × 107 T cells, which has not been reported previously. The present model can be utilized to study human immune cell function in vivo and elucidate the mechanisms underlying the pathogenesis of human GVHD. In addition, this model system can help researchers to rapidly determine whether proposed therapeutic strategies for GVHD are efficient in vivo and will elucidate the underlying mechanisms of drugs and cells to be investigated. Furthermore, such a protocol will undoubtedly be very helpful to laboratories that have no available sources of irradiation.