The function of adipocytes derived from human mesenchymal stem cells (hMSC) was investigated for the first time in hMSC from fetal liver (FL) and adult bone marrow (BM) and compared with preadipocytes from human subcutaneous adipose tissue differentiated according to adipocyte-specific protocols. FL- and BM-derived adipocytes displayed both morphological and functional characteristics of mature adipocytes including specific intracellular signaling pathways for tumor necrosis factor-α, catecholamine-regulated lipolysis as well as secretion of adiponectin and leptin. Similar to differentiated preadipocytes, hMSC adipocytes displayed lipolytic effects mediated by β-adrenoceptors and antilipolytic effects mediated by the α2A-adrenoceptor (α2A-AR) and expressed proteins with a pivotal role in human lipolysis, including β2-AR, α2A-AR, and hormone-sensitive lipase. We conclude that hMSC-derived adipocytes are morphologically and functionally similar to preadipocytes and display an intact lipolytic signaling pathway and endocrine function. These systems could be of great value in adipocyte research as a renewable source of adipocytes.