Mouse 3T3-L1 preadipocytes differentiate into adipocytes when treated with 3-isobutyl-1-methylxanthine, dexamethasone, and insulin. After hormonal induction, growth-arrested 3T3-L1 preadipocytes reenter the cell cycle, known as mitotic clonal expansion, and then express genes required for the adipocyte phenotype. Although the mechanisms of adipogenesis including transcriptional cascade have been revealed during past 40 years, it is still unclear how cells after clonal expansion eventually enter the terminal differentiation program. This study aims to elucidate novel regulators of terminal differentiation after clonal expansion of preadipocytes. When fresh 3T3-L1 preadipocytes were exposed to the conditioned medium harvested from the cells at clonal expansion stage, cells showed acceleration of the differentiation into mature adipocytes, indicating the presence of cell-specific regulators modulating transitional events from mitotic clonal expansion to terminal differentiation. In an effort to identify novel regulators, gene expression profiles of differentiating preadipocytes were analyzed using an algorithm. As a result, I found G0/G1 switch gene 2 (G0s2) as a novel regulator of adipogenesis. G0s2 was expressed at a higher level in white and brown adipose tissues, and induced upon a hormonal cocktail in 3T3-L1 cells. Importantly, G0s2 expression was accelerated by conditioned medium, suggesting that this molecule is associated with the transition into the terminal differentiation. Knockdown of the G0s2 expression using siRNA inhibited adipocyte differentiation, whereas constitutive overexpression of G0s2 accelerated the differentiation from preadipocytes to mature adipocytes. G0s2 expression was regulated by peroxisome proliferator-activated receptor (PPAR) which is the well-known regulator of adipocyte differentiation, and the absence of either PPAR or G0s2 resulted in the activation of apoptotic pathway before undergoing terminal differentiation. To determine whether G0s2 plays a role in vivo, g0s2 knockout mice were generated. G0s2 knockout mice were normal in appearance, except reduction in adipose mass compared with wild-type littermates, indicating an impairment of adipogenesis. These results also suggest that G0s2 plays as a contributing factor in lipid droplet formation. In conclusion, G0s2 is required for adipogenesis and accumulation of triacylglycerol, playing an important role in the transition from mitotic clonal expansion to terminal differentiation.