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Mesenchymal stromal cells (MSCs) have gained special relevance in bone tissue regenerative applications. MSCs have been isolated from different depots, with adipose tissue being acknowledged as one of the most convenient sources, given the wide availability, high cellular yield, and obtainability. Recently, the falciform ligament (FL) has been regarded as a potential depot for adipose tissue-derived stromal cells (FL-ADSCs) isolation. Nonetheless, the osteogenic capability of FL-ADSCs has not been previously characterized. Thus, the present study aimed the detailed characterization of FL-ADSCs' functionality upon osteogenic induction through a classic (dexamethasone-based-DEX) or an innovative strategy with retinoic acid (RA) in a comparative approach with ADSCs from a control visceral region. Cultures were characterized for cell proliferation, metabolic activity, cellular morphology, fluorescent cytoskeletal and mitochondrial organization, and osteogenic activity–gene expression analysis and cytochemical staining. FL-derived populations expressed significantly higher levels of osteogenic genes and cytochemical markers, particularly with DEX induction, as compared to control ADSCs that were more responsive to RA. FL-ADSCs were identified as a potential source for bone regenerative applications, given the heightened osteogenic functionality. Furthermore, data highlighted the importance of the selection of the most adequate osteogenic-inducing program concerning the specificities of the basal cell population. [ABSTRACT FROM AUTHOR]