Cells, scaffolds, and growth factors are the three main factors for creating a stem-cell-based tissue-engineered construct, but the interactions between three factors are not very clear. We hereby explored the interactions between rat-adipose-derived stromal cells (rASCs), recombinant human bone morphogenetic protein-2 (rhBMP-2), and beta-tricalcium phosphate (beta-TCP) to provide evidence for their application in bone tissue engineering by evaluating the protein adsorption of beta-TCP, the cell attachment, alkaline phosphatase (ALP) activity/protein, osteocalcin (OCN) content, mineral formation, calcium content, phosphonium content, cell vitality, gene expression, and implantation in the backs of severe combined immunodeficient mice of rhBMP-2 preinducing rASCs seeded onto beta-TCP. The results showed that beta-TCP could adsorb the proteins from the media. The attachment, proliferation, and osteogenic properties of rASCs were supported by beta-TCP, as revealed using scanning electron microscopy. Compared with rASCs cultured on the culture plate, rASCs cultured on beta-TCP had significantly higher ALP activity/protein, OCN content, and mineral formation. These values for rASCs cultured on beta-TCP with rhBMP-2 increased most significantly. The rhBMP-2 significantly increased the calcium content, phosphonium content, and ALP, type I collagen, and OCN mRNA levels of rASCs cultured on beta-TCP. The methylthiazol tetrazolium method revealed that the vitality of rASCs cultured on beta-TCP with or without rhBMP-2 for 4, 7, and 28 days in vitro was insignificantly different. After 8 and 12 weeks of implantation, each group displayed increased bone formation over the 12-week period. The percentage of the new bone formed areas for beta-TCP/rhBMP-2 and beta-TCP was not significantly different. This value for rASCs/beta-TCP construct was significantly higher than that for beta-TCP group, but the maximal and robust bone formation was presented in rASCs/beta-TCP with rhBMP-2. The results implied that stem cells existed in adult rat adipose tissue. beta-TCP could adsorb rhBMP-2 from the media and had osteoinductivity when alone implanted in the back of severe combined immunodeficient mice. beta-TCP was also sufficient to trigger the differentiation of rASCs toward an osteoblastic phenotype without the addition of osteogenic factor. The rhBMP-2 could better sufficiently induce osteogenic differentiation of rASCs seeded onto beta-TCP. The rASCs and rhBMP-2 could promote the dissolution of beta-TCP to provide Ca2+ and PO4(3-) needed for bone formation. The interactions between the three factors could provide an optimizing microenvironment for osteogenic differentiation of rASCs, and this might be essential for sufficient and timely bone formation in vivo. This study may provide insight into the clinical repair of bone defect with ASCs+beta-TCP+rhBMP-2 construct.