Obesity is characterized by an increase in the number mature fat cells. These nascent adipocytes are derived from preadipocytes, which in turn are derived from mesenchymal stem cells (MSCs). Since little is known about the mechanisms controlling the commitment of MSCs into preadipocytes, this early event in adipogenesis was further investigated. C3H10T1/2 cells (10T1/2 cells) were employed as a MSC model and a committed A33 preadipocyte cell line derived from these cells served as a model of preadipocytes. Microarray technology was used to identify genes that are differentially expressed in pluripotent 10T1/2 cells when compared with A33 preadipocytes. Several key genes of the Wnt signaling pathway were differentially expressed between 10T1/2 and A33 cells as demonstrated by microarray and quantitative real-time RT-PCR analyses. Of particular interest, R-spondins-2 and -3, newly described molecules that activate the canonical Wnt signaling pathway, are markedly upregulated in proliferating A33 cells compared to 10T1/2 cells. Consistent with these findings beta-catenin accumulates in the nuclei of proliferating A33 cells, but not 10T1/2 cells. In addition, several members of the Lef/Tcf family of transcription factors involved in Wnt signaling are also differentially expressed between 10T1/2 and A33 cells. These and other findings indicate that activation of Wnt signaling is an early event in adipogenesis.