Krox20 is a zinc finger-containing transcription factor that is abundantly expressed in adipose tissue. However, its role in fat cell differentiation has not been established. In cultured 3T3-L1 cells, Krox20 is rapidly induced by serum stimulation. Overexpression of Krox20 in both 3T3-L1 preadipocytes and multipotent NIH3T3 cells promotes adipogenesis in a hormone-dependent manner. Conversely, RNAi-mediated loss of Krox20 function reduced adipogenesis in 3T3-L1 cells. Ectopic expression of Krox20 can transactivate the C/EBPbeta promoter and increase C/EBPbeta gene expression in 3T3-L1 preadipocytes. RNAi-mediated knockdown of C/EPBbeta diminished Krox20's proadipogenic effect. Finally, coexpression of Krox20 and C/EBPbeta in naive NIH3T3 cells resulted in the pronounced induction of a fully differentiated adipocyte phenotype, an effect previously observed only with PPARgamma. These data indicate that Krox20 is necessary for adipogenesis and that, when overexpressed, Krox20 potently stimulates adipogenesis via C/EBPbeta-dependent and -independent mechanisms.