The pocket proteins, through their interaction with the E2F transcription factors, ensure the proper regulation of cell proliferation. By doing so, these protein complexes affect other fundamental processes such as differentiation. Here we analyze the in vivo roles of three proteins, pRb, E2F4, and p107, in murine embryonic development and in differentiation in vitro. As Rb loss causes embryonic lethality due to placental defects, we adopted a conditional knockout strategy to generate Rb-deficient embryos that survive to birth. This approach allows us to assess a role for Rb in skeletal development. We find that Rb-inactivation impairs the ossification of several bones. These bone defects correlate with an inability of Rb-deficient osteoblasts to properly exit the cell cycle. Similarly, we find that the mutation of E2f4 causes defects in embryonic ossification that are accompanied by a significantly greater percentage of cycling cells compared to controls. Overall, these findings indicate that both pRb and E2F4 are required in vivo for proper cell cycle arrest of osteoblasts and, consequently, for the normal development of bone. Furthermore, by expanding our analyses to in vitro studies, we show that Rb not only regulates the cell cycle, but also the differentiation properties of the osteoblasts. This correlates with a dramatic upregulation of pro-osteoblastic genes, including Bmp2, Msx2, Runx2 and Osterix. By using the same strategy to conditionally delete Rb in p107/- embryos, we observe lethality at approximately e14.5 during development. Analyzing embryos at e13.5, we find that the combined loss of pRb and p107 affects the development of a variety of organs, including the central nervous system, limbs, blood vessel endothelial cells, and heart. These data demonstrate novel, cooperative functions for pRb and p107 in murine embryogenesis. Altogether, these studies provide evidence of much broader and novel roles for pRb, p107, and E2F4 in the development and differentiation of murine embryonic tissues.