Objective: Graft-vs-host disease (GVHD) is the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Models of immunodeficient mice that consistently and efficiently reconstitute with xenoreactive human T cells would be a valuable tool for the in vivo study of GVHD, as well as other human immune responses. Materials and Methods: We developed a consistent and sensitive model of human GVHD by retro-orbitally injecting purified human T cells into sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID)-β2mnull recipients. In addition, we characterized for the first time the trafficking patterns and expansion profiles of xenoreactive human T cells in NOD/SCID-β2mnull recipients using in vivo bioluminescence imaging. Results: All NOD/SCID-β2mnull mice conditioned with 300 cGy total body irradiation and injected with 1 × 107 human T cells exhibited human T-cell engraftment, activation, and expansion, with infiltration of multiple target tissues and a subsequent >20% loss of pretransplantation body weight. Importantly, histological examination of the GVHD target tissues revealed changes consistent with human GVHD. Furthermore, we also showed by in vivo bioluminescence imaging that development of lethal GVHD in the NOD/SCID-β2mnull recipients was dependent upon the initial retention and early expansion of human T cells in the retro-orbital sinus cavity. Conclusion: Our NOD/SCID-β2mnull mouse model provides a system to study the pathophysiology of acute GVHD induced by human T cells and aids in development of more effective therapies for human GVHD.