There is an urgent and unmet need for humanized in vivo models of type 1 diabetes to study immunopathogenesis and immunotherapy, and in particular antigen-specific therapy. Transfer of patient blood lymphocytes to immunodeficient mice is associated with xenogeneic graft-versus-host reactivity that complicates assessment of autoimmunity. Improved models could identify which human T cells initiate and participate in beta-cell destruction and help define critical target islet autoantigens. We used humanized mice (hu-mice) containing robust human immune repertoires lacking xenogeneic graft-versus-host reactivity to address this question. Hu-mice constructed by transplantation of HLA-DQ8⁺ human fetal thymus and CD34⁺ cells into HLA-DQ8–transgenic immunodeficient mice developed hyperglycemia and diabetes after transfer of autologous HLA-DQ8/insulin-B:9–23 (InsB:9–23)-specific T-cell receptor (TCR)-expressing human CD4⁺ T cells and immunization with InsB:9–23. Survival of the infused human T cells depended on the preexisting autologous human immune system, and pancreatic infiltration by human CD3⁺ T cells and insulitis were observed in the diabetic hu-mice, provided their islets were stressed by streptozotocin. This study fits Koch’s postulate for pathogenicity, demonstrating a pathogenic role of islet autoreactive CD4⁺ T-cell responses in type 1 diabetes induction in humans, underscores the role of the target beta-cells in their immunological fate, and demonstrates the capacity to initiate disease with T cells, recognizing the InsB:9–23 epitope in the presence of islet inflammation. This preclinical model has the potential to be used in studies of the pathogenesis of type 1 diabetes and for testing of clinically relevant therapeutic interventions.