Commensal bacteria produce metabolites that promote the generation of regulatory T (Treg) cells in the periphery. The study shows that short-chain fatty acids (SCFAs), particularly butyrate, produced by gut bacteria enhance extrathymic Treg cell differentiation. Butyrate increases Treg cell numbers by enhancing extrathymic differentiation, which depends on the intronic enhancer CNS1. Propionate also promotes Treg cell generation, while acetate does not. These findings suggest that bacterial metabolites mediate communication between the gut microbiota and the immune system, influencing the balance between pro- and anti-inflammatory responses. Butyrate acts on T cells and dendritic cells (DCs) to enhance Foxp3 expression, a key transcription factor for Treg cells. Butyrate increases histone acetylation, which stabilizes Foxp3 and enhances Treg function. The study also shows that butyrate promotes Treg cell generation in the colon when delivered locally, but not systemically. Other SCFAs, such as acetate and propionate, have different effects on Treg cell generation. The results indicate that bacterial metabolites play a crucial role in maintaining immune homeostasis by modulating Treg cell differentiation. The study used in vitro and in vivo experiments to demonstrate these effects, including the use of SCFA-enriched fecal extracts and butyrate enemas. The findings highlight the importance of the gut microbiota in immune regulation and suggest that modulating SCFA levels could be a therapeutic strategy for immune-related diseases.Commensal bacteria produce metabolites that promote the generation of regulatory T (Treg) cells in the periphery. The study shows that short-chain fatty acids (SCFAs), particularly butyrate, produced by gut bacteria enhance extrathymic Treg cell differentiation. Butyrate increases Treg cell numbers by enhancing extrathymic differentiation, which depends on the intronic enhancer CNS1. Propionate also promotes Treg cell generation, while acetate does not. These findings suggest that bacterial metabolites mediate communication between the gut microbiota and the immune system, influencing the balance between pro- and anti-inflammatory responses. Butyrate acts on T cells and dendritic cells (DCs) to enhance Foxp3 expression, a key transcription factor for Treg cells. Butyrate increases histone acetylation, which stabilizes Foxp3 and enhances Treg function. The study also shows that butyrate promotes Treg cell generation in the colon when delivered locally, but not systemically. Other SCFAs, such as acetate and propionate, have different effects on Treg cell generation. The results indicate that bacterial metabolites play a crucial role in maintaining immune homeostasis by modulating Treg cell differentiation. The study used in vitro and in vivo experiments to demonstrate these effects, including the use of SCFA-enriched fecal extracts and butyrate enemas. The findings highlight the importance of the gut microbiota in immune regulation and suggest that modulating SCFA levels could be a therapeutic strategy for immune-related diseases.