This study was conducted to assess the effects of specific oxysterols on the adipogenic differentiation and expression of adipogenic transcripts in C3H10T1/2 mouse stem cells. In the first study, four oxysterols namely; 20S, 22R, 22S and 25 hydroxycholesterol (25-HC) were tested to determine which one best inhibits adipogenesis in C3H10T1/2 mouse stem cells. Adipogenic differentiation was induced using an adipogenic media (DMITro) consisting of dexamethasone (DEX), 3-isobutyl-1-methyl-xanthine (IBMX), insulin and troglitazone (Tro). Inhibition of adipogenesis was assessed by treatment of cells with DMITro+20S, 22R, 22S or 25-HC for six days. Oil red O pictures and gene expression analysis showed that 25-HC was more effective in inhibiting the expression of adipogenic genes compared to the other oxysterols. Further investigation of the mechanisms of action of 25-HC showed that the inhibitory effects of 25-HC on adipogenesis are not mediated by hedgehog signalling.