CD4+ CD25+ FOXP3+ regulatory T cells (Treg) are pivotal for peripheral self-tolerance. They prevent immune responses to auto-and alloantigens and are thus under close scrutiny as cellular therapeutics for autoimmune diseases and the prevention or treatment of alloresponses after organ or stem cell transplantation. We previously showed that human Treg with a memory cell phenotype, but not those with a naive phenotype, rapidly downregulate expression of the lineage-defining transcription factor FOXP3 upon in vitro expansion. We now compared the transcriptomes of stable FOXP3+ Treg and converted FOXP3− ex-Treg by applying a newly developed intranuclear staining protocol that permits the isolation of intact mRNA from fixed, permeabilized, and FACS-purified cell populations. Whole-genome microarray analysis revealed strong and selective upregulation of Th2 signature genes, including GATA-3, IL-4, IL-5, and IL-13, upon downregulation of FOXP3. Th2 differentiation of converted FOXP3− ex-Treg occurred even under nonpolarizing conditions and could not be prevented by IL-4 signaling blockade. Thus, our studies identify Th2 differentiation as the default developmental program of human Treg after downregulation of FOXP3.