The potential for chemical exposures to exacerbate the development and/or prevalence of metabolic disorders, such as obesity, is currently of great societal concern. Various in vitro assays are available to assess adipocyte differentiation, though little work has been done to standardize protocols and compare models effectively. This study compares several adipogenic cell culture systems under a variety of conditions to assess variability in responses. Two sources of 3T3-L1 preadipocytes as well as OP9 preadipocytes were assessed for cell proliferation and triglyceride accumulation following different induction periods and using various tissue culture plates. Both cell line and cell source had a significant impact on potencies and efficacies of adipogenic chemicals. Gene expression analyses suggested that differential expression of nuclear receptors involved in adipogenesis underlie the differences between OP9 and 3T3-L1 cells; however, there were also differences based on 3T3-L1 cell source. Induction period modulated potency and efficacy of response depending on cell line and test chemical, and large variations were observed in triglyceride accumulation and cell proliferation between brands of tissue culture plates. Our results suggest that the selection of a cell system and differentiation protocol significantly impacts the detection of adipogenic chemicals, and therefore, influences reproducibility of these studies.