Phosphorylated inactive FoxO1, FoxO3a, and FoxO4 were observed in both RA and OA synovial tissue. Inactivation of FoxO1, FoxO3a, and FoxO4 was restricted to FLS, T lymphocytes, and synovial macrophages, respectively. Inactivation of FoxO1 and FoxO4 was significantly enhanced (P < 0.05) in RA synovial tissue compared with OA synovial tissue. No significant differences in the inactivation of T lymphocyte FoxO3a were observed between RA and OA synovial tissue. In vitro, PI3-kinase-dependent inactivation of FoxO1 was observed in RA FLS following stimulation with TNF, TGF, and soluble CD154. Inactivation of FoxO1 was not sufficient to promote FLS proliferation, as only TNF and TGF but not soluble CD154 stimulated PI3-kinase-dependent FLS proliferation. Our studies demonstrate that inactivation of FoxO1 and FoxO4 is enhanced in RA compared with OA, provide the first (patho)physiological evidence of FoxO4 inactivation in vivo, and suggest important roles for FoxO1 and FoxO4 in maintaining, respectively, FLS and macrophage activation and survival in RA synovial tissue.