The phosphatidylinositol 3-kinase (PI3K)/Akt pathway tightly regulates adipose cell differentiation. Here we show that loss of Akt1 /PKB alpha in primary mouse embryo fibroblast (MEF) cells results in a defect of adipocyte differentiation. Adipocyte differentiation in vitro and ex vivo was restored in cells lacking both Akt1/PKB alpha and Akt2/PKB beta by ectopic expression of Akt1/PKB alpha but not Akt2/PKB beta. Akt1/PKB alpha was found to be the major regulator of phosphorylation and nuclear export of Fox01, whose presence in the nucleus strongly attenuates adipocyte differentiation. Differentiation-induced cell division was significantly abrogated in Akt1/PKB alpha-deficient cells, but was restored after forced expression of Akt1/PKB alpha. Moreover, expression of p27(Kip1), an inhibitor of the cell cycle, was down regulated in an Akt1/PKB alpha-specific manner during adipocyte differentiation. Based on these data, we suggest that the Akt1/PKB alpha isoform plays a major role in adipocyte differentiation by regulating Fox01 and p27(Kip1). (C) 2008 Elsevier Inc. All rights reserved.