Human FOXP3 regulatory T (T) cells are central to immune tolerance. However, their heterogeneity and differentiation remain incompletely understood. Here we use single-cell RNA and T cell receptor sequencing to resolve T cells from healthy individuals and patients with or without acute graft-versus-host disease (aGVHD) who undergo stem cell transplantation. These analyses, combined with functional assays, separate T cells into naïve, activated, and effector stages, and resolve the HLA-DR, LIMS1, highly suppressive FOXP3, and highly proliferative MKI67 effector subsets. Trajectory analysis assembles T subsets into two differentiation paths (I/II) with distinctive phenotypic and functional programs, ending with the FOXP3 and MKI67 subsets, respectively. Transcription factors FOXP3 and SUB1 contribute to some Path I and Path II phenotypes, respectively. These FOXP3 and MKI67 subsets and two differentiation pathways are conserved in transplanted patients, despite having functional and migratory impairments under aGVHD. These findings expand the understanding of T cell heterogeneity and differentiation and provide a single-cell atlas for the dissection of T complexity in health and disease.