Much knowledge of adipocyte biology has been learned from cell culture models, most notably 3T3-L1 cells. The 3T3-L1 model has several limitations, including the requirement of 2 weeks to generate adipocytes and the waning of adipogenic potential in culture. We have investigated the capacity of OP9 cells, a line of bone marrow-derived mouse stromal cells, to recapitulate adipogenesis. When OP9 cells are given any one of three adipogenic stimuli, they rapidly accumulate triacylglycerol, assume adipocyte morphology, and express adipocyte late marker proteins, including glucose transporter 4 and adiponectin. OP9 cells can differentiate into adipocytes within 2 days. This rapid rate of differentiation allows for the detection of transiently expressed proteins in mature OP9 adipocytes. Adipogenesis in OP9 cells involves the master transcriptional regulator of adipocyte differentiation, peroxisome proliferator-activated receptor gamma (PPARgamma). OP9 cells are late preadipocytes in that, before the addition of adipogenic stimuli, they express the adipocyte proteins CCAAT/enhancer binding proteins alpha and beta, PPARgamma, sterol-regulatory element binding protein-1, S3-12, and perilipin. OP9 differentiation is not diminished by maintenance in culture at high cell density or by long periods in continuous culture, thereby facilitating the generation of stable cell lines that retain adipogenic potential. Thus, the unique features of OP9 cells will expedite the study of adipocyte biology.