The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-beta2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP)alpha and C/EBPbeta alpha at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor gamma (PPARgamma2) transcripts at 7 days. TGF-beta2 administration in unloaded rats corrected the rise in C/EBPalpha and C/EBPbeta transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPARgamma2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBPalpha and C/EBPbeta expression by TGF-beta2 was associated with increased PPARgamma serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPARgamma transactivating activity. The sequential inhibitory effect of TGF-beta2 on C/EBPalpha, C/EBPbeta, and PPARgamma2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-beta2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBPalpha, C/EBPbeta, and PPARgamma expression and activity, which provides a sequential mechanism by which TGF-beta2 regulates adipogenic differentiation of bone marrow stromal cells in vivo.