AbstractIntroduction: Ephrin‐B2 on osteoclasts was reported to promote bone formation as part of homeostasis by activating the EphB4 tyrosine kinase receptor on osteoblasts. Little is known about the role of ephrin‐B signaling to EphBs in developmental bone formation. Results: We observed expression of an ephrin‐B2 LacZ chimeric allele in the periosteum, sutural bone fronts, and dura mater of embryonic and neonatal mice. Expression in the adult skull was confined to sutures, but was heavily upregulated at sites of bone injury. Culture of embryonic calvariae with soluble recombinant ephrin‐B2/Fc doubled their bone content without altering suture width or overall skull morphology. Ephrin‐B2/Fc also stimulated osteoblast marker gene expression in cultured MC3T3 preosteoblastic cells without the need for type 1 collagen‐induced differentiation. EphB4 was absent in embryonic and adult skulls. However, EphB1 and EphB2, both physiological receptors for ephrin‐Bs, were expressed at sites of osteogenesis, and EphB1 knockout mice displayed a reduction in calvarial bone content compared to controls. Conclusions: These data support a role for ephrin‐B2 in the development and healing of bone through activation of osteoblast‐specific gene expression. EphB1 and EphB2 are likely candidates receptors for the ephrin‐B2 in bone. Developmental Dynamics, 2012. © 2012 Wiley Periodicals, Inc.