Forkhead winged-helix transcription factor Foxp3 serves as the dedicated mediator of the genetic program governing CD25 ⁺ CD4 ⁺ regulatory T cell (T _r ) development and function in mice. In humans, its role in mediating T _r development has been controversial. Furthermore, the fate of T _r precursors in FOXP3 deficiency has yet to be described. Making use of flow cytometric detection of human FOXP3, we have addressed the relationship between FOXP3 expression and human T _r development. Unlike murine Foxp3 ⁻ T cells, a small subset of human CD4 ⁺ and CD8 ⁺ T cells transiently up-regulated FOXP3 upon in vitro stimulation. Induced FOXP3, however, did not alter cell-surface phenotype or suppress T helper 1 cytokine expression. Furthermore, only ex vivo FOXP3 ⁺ T _r cells persisted after prolonged culture, suggesting that induced FOXP3 did not activate a T _r developmental program in a significant number of cells. FOXP3 flow cytometry was also used to further characterize several patients exhibiting symptoms of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) with or without FOXP3 mutations. Most patients lacked FOXP3-expressing cells, further solidifying the association between FOXP3 deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Interestingly, one patient bearing a FOXP3 mutation enabling expression of stable FOXP3 ^mut protein exhibited FOXP3 ^mut -expressing cells among a subset of highly activated CD4 ⁺ T cells. This observation raises the possibility that the severe autoimmunity in FOXP3 deficiency can be attributed, in part, to aggressive T helper cells that have developed from T _r precursors.