The 3T3-L1 model of in vitro adipogenesis has provided key insights into the molecular nature of this process. However, given that 3T3-L1 are of an embryonic origin, it is not clear to what extent they represent adipogenesis as it occurs in white adipose tissue (WAT). With the goal of better defining preadipocytes and adipogenesis in WAT we have generated a new cell culture model from adipocyte precursors present in C57BL/6 mouse subcutaneous WAT. ScAP-23 preadipocytes show fibroblastic morphology and upon treatment with dexamethasone, 3-methyl-1-isobutylxanthine, insulin and indomethacin, convert to nearly 100% adipocyte morphology. ScAP-23 adipocytes contain abundant lipid droplets and express transcripts for PPAR, C/EBP family and SREBP-1c transcription factors, SCD1, aFABP, ATGL, GLUT4, FAS, LDL, GPDH, and are insulin-responsive. Differential screening of 1,176 genes using nylon DNA arrays identified 10 transcripts enriched in ScAP-23 adipocytes vs. preadipocytes and 26 transcripts enriched in ScAP-23 preadipocytes vs. adipocytes. Semiquantitative or real-time PCR analyses identified a common cohort of 14 transcripts markedly downregulated in both ScAP-23 and 3T3-L1 adipogenesis. These included catenin 1, chemokine ligand 2, serine or cysteine peptidase inhibitor f1, aurora kinase B, thrombospondin2, and solute carrier 7a5. Five of these transcripts (Ccl2, Serpinf1, Aurkb, Thbs2, and Slc7a5) demonstrated at least a 2-fold increase in WAT from obese mice compared to that of wild type mice. This suggests that comparative gene expression studies of ScAP-23 and 3T3-L1 adipogenesis may be particularly fruitful in identifying preadipocyte-expressed genes that play a role in adipose tissue physiology and/or pathophysiology.