Understanding the heterogeneity of human CD4⁺FOXP3⁺ regulatory T cells (Tregs) and their potential for lineage reprogramming is of critical importance for moving Treg therapy into the clinics. Using multiparameter single-cell analysis techniques, we explored the heterogeneity and functional diversity of human Tregs in healthy donors and in patients after allogeneic hematopoietic stem cell transplantation (alloHSCT). Human Tregs displayed a level of complexity similar to conventional CD4⁺ effector T cells with respect to the expression of transcription factors, homing receptors and inflammatory cytokines. Single-cell profiling of the rare Treg producing interleukin-17A or interferon-γ showed an overlap of gene expression signatures of Th17 or Th1 cells and of Tregs. To assess whether Treg homeostasis is affected by an inflammatory and lymphopenic environment, we characterized the Treg compartment in patients early after alloHSCT. This analysis suggested a marked depletion of Treg with a naive phenotype in patients developing acute graft-versus-host disease, compared with tolerant patients. However, single-cell profiling showed that CD4⁺FOXP3⁺ T cells maintain the Treg gene expression signature and Treg-suppressive activity was preserved. Our study establishes that heterogeneity at the single-cell level, rather than lineage reprogramming of CD4⁺FOXP3⁺ T cells, explains the remarkable complexity and functional diversity of human Tregs.