To obtain a molecular definition of regulatory T (Treg) cell identity, we performed proteomics and transcriptomics on various populations of human regulatory and conventional CD4⁺ T (Tconv) cells. A protein expression signature was identified that defines all Treg cells, and another signature that defines effector Treg cells. These signatures could not be extrapolated from transcriptome data. Unique cell-biological and metabolic features in Treg cells were defined, as well as specific adaptations in cytokine, TCR, and costimulatory receptor signaling pathways. One such adaptation—selective STAT4 deficiency—prevented destabilization of Treg cell identity and function by inflammatory cytokines, while these signals could still induce critical transcription factors and homing receptors via other pathways. Furthermore, our study revealed surface markers that identify FOXP3⁺CD4⁺ T cells with distinct functional properties. Our findings suggest that adaptation in signaling pathways protect Treg cell identity and present a resource for further research into Treg cell biology.