Studies of the monogenic autoimmune disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) have elucidated the essential function of the transcription factor FOXP3 and thymic-derived regulatory T cells (T _regs ) in controlling peripheral tolerance. However, the presence and the source of autoreactive T cells in IPEX remain undetermined. Here, we investigated how FOXP3 deficiency affects the T cell receptor (TCR) repertoire and T _reg stability in vivo and compared T cell abnormalities in patients with IPEX with those in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). To study T _regs independently of their phenotype and to analyze T cell autoreactivity, we combined T _reg -specific demethylation region analyses, single-cell multiomic profiling, and bulk TCR sequencing. We found that patients with IPEX, unlike patients with APECED, have expanded autoreactive T cells originating from both autoreactive effector T cells (T _effs ) and T _regs . In addition, a fraction of the expanded T _regs from patients with IPEX lost their phenotypic and functional markers, including CD25 and FOXP3. Functional experiments with CRISPR-Cas9–mediated FOXP3 knockout T _regs and T _regs from patients with IPEX indicated that the patients’ T _regs gain a T _H 2-skewed T _eff -like function, which is consistent with immune dysregulation observed in these patients. Analyses of FOXP3 mutation-carrier mothers and a patient with IPEX after hematopoietic stem cell transplantation indicated that T _regs expressing nonmutated FOXP3 prevent the accumulation of autoreactive T _effs and unstable T _regs . These findings could be directly used for diagnostic and prognostic purposes and for monitoring the effects of immunomodulatory treatments.