Compelling evidence demonstrates that intestinal commensal microbiota modulate conventional and regulatory T cell (Treg) responses that are required for effective host defence against pathogens and avoidance of autoimmunity and other immunopathologic conditions. Here, we investigated the contribution of the commensal microbiota and Toll-like receptor (TLR) signaling to homeostasis of Foxp3 ⁻ conventional CD4 ⁺ T cells and Foxp3 ⁺ Tregs. Upon long-term antibiotics treatment, we observed a significant reduction of conventional CD4 ⁺ T cell proliferation in a systemic manner, whereas Foxp3 ⁺ Treg proliferation was locally impaired in gut-draining mesenteric lymph nodes and Peyer's patches. The proliferative response to microbial components was not mediated by TLRs as MyD88- and various TLR-deficient mice displayed normal or even increased conventional T cell and Foxp3 ⁺ Treg proliferation. Thus, commensal microbiota-derived stimuli support cycling of both conventional CD4 ⁺ T cells and Foxp3 ⁺ Tregs with TLR-mediated recognition of bacterial components not being the major mechanism controlling microbiota-driven T cell homeostasis.