The differentiation of preadipocytes to adipocytes is orchestrated by the expression of the "master adipogenic regulators," CCAAT/enhancer-binding protein (C/EBP) beta, peroxisome proliferator-activated receptor gamma (PPARgamma), and C/EBP alpha. In addition, activation of the cAMP-response element-binding protein (CREB) is necessary and sufficient to promote adipogenic conversion and prevent apoptosis of mature adipocytes. In this report we used small interfering RNA to deplete CREB and the closely related factor ATF1 to explore the ability of the master adipogenic regulators to promote adipogenesis in the absence of CREB and probe the function of CREB in late stages of adipogenesis. Loss of CREB/ATF1 blocked adipogenic conversion of 3T3-L1 cells in culture or 3T3-F442A cells implanted into athymic mice. Loss of CREB/ATF1 prevented the expression of PPARgamma, C/EBP alpha, and adiponectin and inhibited the loss of Pref-1. Loss of CREB/ATF1 inhibited adipogenic conversion even in cells ectopically expressing C/EBP alpha, C/EBP beta, or PPARgamma2 individually. CREB/ATF1 depletion did not attenuate lipid accumulation in cells expressing both PPARgamma2 and C/EBP alpha, but adiponectin expression was severely diminished. Conversely ectopic expression of constitutively active CREB overcame the blockade of adipogenesis due to depletion of C/EBP beta but not due to loss of PPARgamma2 or C/EBP alpha. Depletion of CREB/ATF1 did not suppress the expression of C/EBP beta as we had previously observed using dominant negative forms of CREB. Finally results are presented showing that CREB promotes PPARgamma2 gene transcription. The results indicate that CREB and ATF1 play a central role in adipogenesis because expression of individual master adipogenic regulators is unable to compensate for their loss. The data also indicate that CREB not only functions during the initiation of adipogenic conversion but also at later stages.