Mesenchymal stromal cells (MSCs) in bone marrow are important for bone homeostasis. Although platelet-derived growth factor (PDGF) has been reported to be involved in osteogenic differentiation of MSCs, the role remains controversial and the network of PDGF signaling for MSCs has not been clarified. To clarify the underlying regulatory mechanism of MSC functions mediated by PDGF, we deleted the PDGF receptor (PDGFR)β gene by Cre-loxP strategy and examined the role of PDGF in osteogenic differentiation of MSCs and fracture repair. In cultured MSCs, the mRNA expression of PDGF-A, -B, -C, and -D as well as PDGFRα and β was detected. Depletion of PDGFRβ in MSCs decreased the mitogenic and migratory responses and enhanced osteogenic differentiation as evaluated by increased alkaline phosphatase (ALP) activity and mRNA levels of ALP, osteocalcin (OCN), bone morphogenetic protein (BMP) 2, Runx2, and osterix in quantitative RT-PCR. PDGF-BB, but not PDGF-AA, inhibited osteogenic differentiation accompanied by decreased ALP activity and mRNA levels, except for BMP2. These effects of PDGF-BB were eliminated by depletion of PDGFRβ in MSCs except that PDGF-BB still suppressed osterix expression in PDGFRβ-depleted MSCs. Depletion of PDGFRβ significantly increased the ratio of woven bone to callus after fracture. From the combined analyses of PDGF stimulation and specific PDGFRβ gene deletion, we showed that PDGFRβ signaling distinctively induces proliferative and migratory responses but strongly inhibits osteogenic differentiation of MSCs. The effects of PDGFRα on the osteogenic differentiation were very subtle. PDGFRβ could represent an important target for guided tissue regeneration or tissue engineering of bone.