A screen of microRNAs preferentially expressed in osteoblasts identified members of the miR-34 family as regulators of osteoblast proliferation and/or differentiation. Osteoblast-specific gain- and loss-of-function experiments performed in vivo revealed that miR-34b and -c affected skeletogenesis during embryonic development, as well as bone mass accrual after birth, through two complementary cellular and molecular mechanisms. First, they inhibited osteoblast proliferation by suppressing Cyclin D1, CDK4, and CDK6 accumulation. Second, they inhibited terminal differentiation of osteoblasts, at least in part through the inhibition of SATB2, a nuclear matrix protein that is a critical determinant of osteoblast differentiation. Genetic evidence obtained in the mouse confirmed the importance of SATB2 regulation by miR-34b/c. These results are the first to identify a family of microRNAs involved in bone formation in vivo and to identify a specific genetic pathway by which these microRNAs regulate osteoblast differentiation.