AbstractHuman FOXP3⁺ regulatory T (T_reg) 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_reg 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_reg cells into naïve, activated, and effector stages, and resolve the HLA-DR^hi, LIMS1^hi, highly suppressive FOXP3^hi, and highly proliferative MKI67^hi effector subsets. Trajectory analysis assembles T_reg subsets into two differentiation paths (I/II) with distinctive phenotypic and functional programs, ending with the FOXP3^hi and MKI67^hi subsets, respectively. Transcription factors FOXP3 and SUB1 contribute to some Path I and Path II phenotypes, respectively. These FOXP3^hi and MKI67^hi 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_reg cell heterogeneity and differentiation and provide a single-cell atlas for the dissection of T_reg complexity in health and disease.