Regulatory T (Treg) cells are essential for maintaining immune homeostasis by suppressing immune-mediated inflammation and autoimmunity. The discovery of Foxp3 as the transcription factor specifying the Treg lineage has advanced understanding of Treg biology. Treg cells differentiate in the thymus and periphery, with thymic Treg cells (tTreg) arising from self-reactive T cells that undergo negative selection, while peripheral Treg cells (iTreg) develop in response to non-self antigens. TCR signaling, particularly high-affinity interactions with self-peptide-MHC complexes, is crucial for tTreg differentiation, while iTreg differentiation requires suboptimal costimulation and TGF-β signaling. IL-2 and TGF-β are essential for Foxp3 induction in peripheral T cells, with TGF-β playing a key role in iTreg generation. Akt activation and PI3K-mTOR signaling also influence Treg differentiation, with sustained Akt activity inhibiting Foxp3 induction. Treg cells are primarily of thymic origin, with peripheral Treg pools showing significant overlap in TCR repertoires with thymic Treg cells. Mucosal tissues, such as gut-associated lymphoid tissues (GALT), provide an environment conducive to iTreg generation due to the presence of CD103+ dendritic cells and exposure to commensal microbiota. These tissues support iTreg differentiation through antigen presentation, TGF-β, and retinoic acid signaling. Treg cells regulate immune responses to environmental antigens and prevent excessive inflammation, highlighting their critical role in maintaining immune tolerance.Regulatory T (Treg) cells are essential for maintaining immune homeostasis by suppressing immune-mediated inflammation and autoimmunity. The discovery of Foxp3 as the transcription factor specifying the Treg lineage has advanced understanding of Treg biology. Treg cells differentiate in the thymus and periphery, with thymic Treg cells (tTreg) arising from self-reactive T cells that undergo negative selection, while peripheral Treg cells (iTreg) develop in response to non-self antigens. TCR signaling, particularly high-affinity interactions with self-peptide-MHC complexes, is crucial for tTreg differentiation, while iTreg differentiation requires suboptimal costimulation and TGF-β signaling. IL-2 and TGF-β are essential for Foxp3 induction in peripheral T cells, with TGF-β playing a key role in iTreg generation. Akt activation and PI3K-mTOR signaling also influence Treg differentiation, with sustained Akt activity inhibiting Foxp3 induction. Treg cells are primarily of thymic origin, with peripheral Treg pools showing significant overlap in TCR repertoires with thymic Treg cells. Mucosal tissues, such as gut-associated lymphoid tissues (GALT), provide an environment conducive to iTreg generation due to the presence of CD103+ dendritic cells and exposure to commensal microbiota. These tissues support iTreg differentiation through antigen presentation, TGF-β, and retinoic acid signaling. Treg cells regulate immune responses to environmental antigens and prevent excessive inflammation, highlighting their critical role in maintaining immune tolerance.