ABSTRACT   Cbfb is a cotranscription factor that forms a heterodimer with Runx proteins Runx1, Runx2, and Runx3. It is required for fetal liver hematopoiesis and skeletal development. Cbfb has two functional isoforms, Cbfb1 and Cbfb2, which are formed by alternative splicing. To address the biological functions of these isoforms in skeletal development, we examined Cbfb1–/– and Cbfb2–/– mouse embryos. Intramembranous and endochondral ossification was retarded and chondrocyte and osteoblast differentiation was inhibited in Cbfb2–/– embryos but not in Cbfb1–/– embryos. Cbfb2 mRNA was upregulated in calvariae, limbs, livers, thymuses, and hearts of Cbfb1–/– embryos but Cbfb1 mRNA was not in those of Cbfb2–/– embryos, and the total amount of Cbfb1 and Cbfb2 mRNA in Cbfb1–/– embryos was similar to that in wild-type embryos but was severely reduced in Cbfb2–/– embryos. The absolute numbers of Cbfb2 mRNA in calvariae, limbs, livers, thymuses, and brains in wild-type embryos were about three times higher than those of Cbfb1 in the respective tissue. The levels of Runx proteins were reduced in calvariae, limbs, and primary osteoblasts from Cbfb2–/– embryos, but the reduction in Runx2 protein was very mild. Furthermore, the amounts of Runx proteins and Cbfb in Cbfb2–/– embryos differed similarly among skeletal tissues, livers, and thymuses, suggesting that Runx proteins and Cbfb are mutually required for their stability. Although Cbfb1–/– embryos developed normally, Cbfb1 induced chondrocyte and osteoblast differentiation and enhanced DNA binding of Runx2 more efficiently than Cbfb2. Our results indicate that modulations in the relative levels of the isoforms may adjust transcriptional activation by Runx2 to appropriate physiological levels. Cbfb2 was more abundant, but Cbfb1 was more potent for enhancing Runx2 activity. Although only Cbfb2 loss generated overt skeletal phenotypes, both may play major roles in skeletal development with functional redundancy. © 2016 American Society for Bone and Mineral Research.