Glucocorticoids provide an adipogenic stimulus that is most obvious in the truncal obesity of patients with Cushing’s syndrome. Glucocorticoid treatment also strongly potentiates the differentiation of human preadipocytes in culture. However, the molecular basis of these stimulatory effects remains to be defined. In this study we provide a detailed analysis of the specific contribution of glucocorticoid treatment to the differentiation of primary human preadipocytes cultured in chemically defined medium. Contrary to previous descriptions of glucocorticoids being required throughout the course of differentiation, our results show that glucocorticoid treatment is only stimulatory during the first 48 h of differentiation. Further, stimulation by glucocorticoids and the PPAR agonist troglitazone is mediated sequentially. Several details of the early events in the differentiation of human preadipocytes and the contribution of steroid to these events differ from the responses observed previously in murine preadipocyte models. First, glucocorticoid treatment stimulated the early accumulation of C/EBPin primary human preadipocytes. Second, induction of C/EBPin primary human preadipocytes was noted within 4 h of adipogenic stimulus, whereas C/EBP induction is not detected until 24-48 h in the murine 3T3 L1 preadipocyte model. Remarkably, by contrast to human primary preadipocytes which do not undergo post-confluent mitosis, 3T3 L1 murine preadipocytes stimulated to differentiate under chemically defined conditions required glucocorticoids to survive the clonal expansion that precedes terminal differentiation revealing a novel signal imparted by glucocorticoids in this immortalized murine cell system.