Bone formation and remodeling require continuous generation of osteoprogenitor cells from bone marrow stromal cells (MSC), which generate and respond to a variety of growth factors with putative roles in hematopoiesis and mesenchymal differentiation. In this study we examine the interaction of two such factors on the maturation of skeletal components. We previously reported that these factors, hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D(3) (vitD(3)), act together to increase alkaline phosphatase in chondroblasts. We now describe the cooperative effect of these agents on MSC isolated and cultured from human vertebral bone marrow. MSC (passages 3-9) isolated from bone marrow cells of human vertebrae (T1-L5) from 22-36-year-old normal donors were first expanded in vitro and then plated in the presence or absence of 10 ng/mL HGF and/or 10 nmol/L vitD(3), for 7-18 days. HGF treatment increased cell proliferation 2.5-fold, with no effect on alkaline phosphatase activity. Whereas vitD(3) treatment inhibited cell growth by 50%, alkaline phosphatase activity was stimulated eightfold, although no mineralization was observed. HGF together with vitD(3) increased cell proliferation 1.5-fold and alkaline phosphatase activity 13-fold over untreated control. Moreover, mineralization was detected only with this combination. Our findings provide evidence that HGF in concert with vitamin D may promote growth and differentiation of human MSC into osteogenic cells.