Lipomas are benign adipose tissue tumors of unknown etiology, which can vary in size, number, body localization and cell populations within the tissue. Lipoma-derived stem cells (LDSCs) are proposed as a potential tool in regenerative medicine and tissue engineering due to their similar characteristics with adipose-derived stem cells (ADSCs) reported so far. Our study is among the first giving detailed insights into the molecular signature and differences in the differentiation capacity of LDSCs in vitro compared to ADSCs. Mesenchymal stem cell phenotype was analyzed by gene expression and flow cytometric analysis of stem cell markers. Adipogenesis and osteogenesis were analyzed by microscopic analysis, cytochemical and immunocytochemical staining, gene and protein expression analyses. We showed that both LDSCs and ADSCs were mesenchymal stem cells with similar phenotype and stemness state but different molecular basis for potential differentiation. Adipogenesis-related genes expression pattern and presence of more mature adipocytes in ADSCs than in LDSCs after 21 days of adipogenic differentiation, indicated that differentiation capacity of LDSCs was significantly lower compared to ADSCs. Analysis of osteogenesis-related markers after 16 days of osteogenic differentiation revealed that both types of cells had characteristic osteoblast-like phenotype, but were at different stages of osteogenesis. Differences observed between LDSCs and ADSCs are probably due to the distinct molecular signature and their commitment in the tissue that governs their different capacity and fate during adipogenic and osteogenic induction in vitro despite their similar mesenchymal phenotype.