We studied of osteogenic differentiation of multipotent mesenchymal stromal cells from human adipose tissue. Experiments showed that 1α,25-dihydroxycalciferol is a more effective inductor of osteogenesis than dexamethasone. Comparative analysis revealed activation of gene expression for the major osteogenic markers on day 7 of culturing in a medium containing 1α,25-dihydroxycalciferol. It was found that transcription of genes encoding type 1 collagen proteins, osteopontin, osteocalcin, and bone sialoprotein peaked on day 14 in culture, while the expression of alkaline phosphatase and bone morphogenetic protein-2 genes increased over 21 days. Intensive mineralization of the extracellular matrix was observed starting from day 14 in culture. On the basis of the analysis of these data, optimal terms for osteogenic induction (day 14) and an optimal inductor (1α,25-dihydroxycalciferol) were chosen and the protocol of effective osteogenic differentiation of multipotent mesenchymal stromal cells from human adipose tissue was developed for creation of tissue-engineered bone equivalents.