AbstractRunx2 is an essential transcription factor for osteoblast development from mesenchymal progenitors. Runx2 regulates gene expression by interacting with numerous transcription factors and co‐activators to integrate signaling events within the nucleus. In this study we used affinity purification and proteomic techniques to identify novel Runx2 interacting proteins. One of these proteins is the DEAD box RNA helicase, p68 (Ddx5). p68 regulates many aspects of RNA expression, including transcription and splicing. p68 co‐localized with Runx2 in punctate foci within the nucleus. In transcription assays, p68 functioned as a co‐activator of Runx2, but its helicase activity was not essential for co‐activation. In accordance, Runx2 transcriptional activity was muted in p68‐suppressed cells. Surprisingly, osteoblast differentiation of the multipotent progenitor C2C12 cell line was accelerated by p68 suppression and Runx2 suppressed p68 expression in calvarial progenitor cells. Together these data demonstrate that p68 is a novel co‐activator for Runx2, but it inhibits osteogenic differentiation of progenitor cells. Moreover Runx2 has an active role in regulating p68 levels in osteoblast precursors. Thus, crosstalk between Runx2 and p68 controls osteoblast specification and maturation at multiple levels. J. Cell. Biochem. 103: 1438–1451, 2008. © 2007 Wiley‐Liss, Inc.