Peroxisome proliferator-activated receptor-gamma (PPARgamma) is essential for adipogenesis. Since EDF-1 is a cofactor of PPARgamma, we investigated the molecular cross-talk between EDF-1 and PPARgamma in adipogenesis. While EDF-1 was not modulated during differentiation of 3T3-L1 cells, it co-immunoprecipitated with PPARgamma. Silencing EDF-1 by shRNAs inhibited the differentiation in adipocytes of 3T3-L1 cells, as detected by the staining of intracellular triglycerides and the expression of the PPARgamma target gene aP2. Accordingly, we found that anti-EDF-1 shRNAs decreased ligand dependent activation of PPARgamma in 3T3-L1 transiently transfected with a vector expressing luciferase under the control of a PPARgamma responsive consensus. To rule out that this inhibition is due to the concomitant downregulation of PPARgamma levels, we overexpressed PPARgamma in 3T3-L1 silencing EDF-1 and found a decrease of ligand dependent activation of PPARgamma, in spite of the high amounts of PPARgamma. These results demonstrate that EDF-1 is required for PPARgamma transcriptional activation during 3T3-L1 differentiation.