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Summary: Primary human bone marrow adipocytes (BM-Ads) display a specific metabolism that is not recapitulated by in vitro differentiated bone marrow mesenchymal stromal cells. These findings highlight the need for using primary BM-Ads in studies of the metabolic impact of BM-Ads on surrounding cells. Here, we present a protocol for isolating human BM-Ads from bone marrow aspirates and verifying adipocyte suspension purity. These isolated and purified BM-Ads can be used for functional assays or frozen for molecular analyses.For complete details on the use and execution of this protocol, please refer to Attane et al. (2020).