Adipocytes are terminally differentiated cells derived from fibroblastic preadipocyte precursors. Here, we describe a method for the isolation and proliferation of preadipocytes from murine subcutaneous white adipose tissue, followed by differentiation in culture to mature adipocytes; we refer to these cells as primary preadipocytes differentiated in vitro (PPDIVs). Compared to adipogenic cell lines, PPDIV metabolism and adipokine secretion more closely resemble in vivo adipocyte biology. While primary mature adipocytes have the greatest in vivo relevance, their fragility and buoyancy make them unsuitable for many cell culture-based methods. PPDIVs can also take advantage of transgenic and knockout mouse models to produce genetically modified adipocytes. Thus, PPDIVs are a valuable resource for studying adipocyte biology in cell culture.