In vitro differentiation of the progenitor cells or preadipocytes into adipocytes is usually achieved by adding an adipogenic mixture (isobutylmethylxanthine, dexamethasone, and insulin, IDI) to medium supplemented with fetal bovine serum (FBS). To study the effects of steroid hormones in vitro, endogenous hormones, growth factors and cytokines are removed by charcoal stripping of serum. However, the effects of charcoal-stripped serum (CS-FBS) per se on adipogenesis have been ignored. Here, we showed that alkaline phosphate activity and nodule formation of osteoprogenitor KS483 cells were lower in CS-FBS than in FBS. Concurrently, abundant amounts of adipocytes were only observed in KS483 cells cultured with CS-FBS, irrespective of the brands of serum used. Inhibition of the p42/44 MAPK pathway by its specific inhibitor PD98059 increased adipogenesis of KS483 cells with FBS, whereas activation of this signalling pathway by EGF blocked adipogenesis of these cells with CS-FBS. Furthermore, the p42/44 MAPK phosphorylation of KS483 cells cultured with CS-FBS was decreased compared with FBS. We concluded that charcoal-stripping of serum removed stimulators of the MAPK signalling pathway and in turn led to downregulation of osteogenesis and upregulation of adipogenesis. Interestingly, the adipogenic mixture IDI stimulated adipogenesis of KS483 cells cultured with CS-FBS, but not with FBS. Furthermore, differential effects of genistein on adipogenesis were observed in KS483 cells cultured with FBS or CS-FBS in combination with IDI. Our results showed that charcoal stripping of serum affected the commitment of KS483 cells and therefore differentially regulated adipogenesis influenced by IDI alone and in combination with genistein.