Human adipose tissues surgically resected from the subcutaneous abdominal region were enzymatically processed to obtain Human Adipose Stem cells (fibroblast-like adipose tissue-derived stromal cells-ADSC-FL) that were immunophenotypically characterized using a panel of mesenchymal markers by flow cytometry. The formation of new hydroxyapatite crystals in culture dishes, by differentiating cells, further demonstrate the osteogenic potential of purified cells. The aim of this study was to evaluate the osteogenic differentiation potential of ADSC-FL seeded onto a porous beta-tricalcium phosphate (beta-TCP) matrix. ADSC-FL was cultured on the beta-TCP matrix in medium with or without osteogenic differentiation additives. Time-dependent cell differentiation was monitored using osteogenic markers such as alkaline phosphatase (activity assay), osteocalcin and ostopontin (ELISA method) expression. Our results reveal that beta-TCP triggers the differentiation of ADSC-FL toward an osteoblastic phenotype irrespective of whether the cells are grown in a proliferative or a differentiative medium. Hence, a beta-TCP matrix is sufficient to promote osteoblastic differentiation of ADSC-FL. However, in proliferative medium, alkaline phosphatase activity was detected at lower level respect to differentiative medium and osteocalcin and osteopontin showed an expression delay in cells cultured in proliferative medium respect to differentiative one. Moreover, we observed an increase in FAK phosphorylation at level of tyrosine residue in position 397 (Western-blot) that indicates a good cell adhesion to beta-TCP scaffold. In conclusion, our paper demonstrates that a three-dimensional beta-TCP scaffold in vitro triggers on its own the differentiation of ADSC-FL toward an osteoblastic phenotype without the need to use differentiative media.