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Summary: White adipose tissue (WAT) is a central factor in the development of type 2 diabetes, but there is a paucity of translational models to study mature adipocytes. We describe a method for the culture of mature white adipocytes under a permeable membrane. Compared to existing culture methods, MAAC (membrane mature adipocyte aggregate cultures) better maintain adipogenic gene expression, do not dedifferentiate, display reduced hypoxia, and remain functional after long-term culture. Subcutaneous and visceral adipocytes cultured as MAAC retain depot-specific gene expression, and adipocytes from both lean and obese patients can be cultured. Importantly, we show that rosiglitazone treatment or PGC1α overexpression in mature white adipocytes induces a brown fat transcriptional program, providing direct evidence that human adipocytes can transdifferentiate into brown-like adipocytes. Together, these data show that MAAC are a versatile tool for studying phenotypic changes of mature adipocytes and provide an improved translational model for drug development. : Mature adipocytes are notoriously difficult to culture. Here, Harms et al. describe a robust method for the long-term culture of mature white adipocytes under permeable membranes, which preserves adipocyte identity and function. Using this approach, they also show that human mature white adipocytes can transdifferentiate into brown-like adipocytes. Keywords: adipocyte, white adipose, WAT, brown adipose, BAT, transdifferentiation, UCP1, culture, MAAC