Endochondral ossification consists of successive steps of chondrocyte differentiation, including mesenchymal condensation, differentiation of chondrocytes, and hypertrophy followed by mineralization and ossification. Loss-of-function studies have revealed that abnormal growth plate cartilage of the Cdc42 mutant contributes to the defects in endochondral bone formation. Here, we have investigated the roles of Cdc42 in osteogenesis and signaling cascades governing Cdc42-mediated chondrogenic differentiation. Though deletion of Cdc42 in limb mesenchymal progenitors led to severe defects in endochondral ossification, either ablation of Cdc42 in limb preosteoblasts or knockdown of Cdc42 in vitro had no obvious effects on bone formation and osteoblast differentiation. However, in Cdc42 mutant limb buds, loss of Cdc42 in mesenchymal progenitors led to marked inactivation of p38 and Smad1/5, and in micromass cultures, Cdc42 lay on the upstream of p38 to activate Smad1/5 in bone morphogenetic protein-2-induced mesenchymal condensation. Finally, Cdc42 also lay on the upstream of protein kinase B to transactivate Sox9 and subsequently induced the expression of chondrocyte differential marker in transforming growth factor-b1-induced chondrogenesis. Taken together, by using biochemical and genetic approaches, we have demonstrated that Cdc42 is involved not in osteogenesis but in chondrogenesis in which the BMP2/Cdc42/Pak/p38/Smad signaling module promotes mesenchymal condensation and the TGF-b/Cdc42/Pak/Akt/Sox9 signaling module facilitates chondrogenic differentiation. KEYWORDS Cdc42; condensation; osteoblast; chondrocyte S EVERAL successive steps, including mesenchymal condensation of undifferentiated cells, chondrocyte differentiation, proliferation of chondrocytes, and differentiation into hypertrophic chondrocytes followed by mineralization and ossification, exist in the process of endochondral ossification (Long and Ornitz 2013). Mesenchymal condensation is a prerequisite for chondrogenesis and is facilitated by cell adhesion molecules. Upregulation of cell adhesion proteins such as N-cadherin is a hallmark of condensing cells, whereas loss of cell adhesion molecules is able to abolish condensation (Delise and Tuan 2002;Bobick et al. 2009). The molecular mechanisms governing condensation are not fully understood, though several genes have been implicated in this process such as bone morphogenic proteins (BMPs) and SRY (sex determining region Y)-box 9 (Sox9) (Fromental-Ramain et al. 1996;Wada et al. 1998;Lu et al. 2008). Conditional inactivationof Sox9 in limb mesenchymal progenitors leads to the absence of condensations, while simultaneous deletion of Bmp2 and Bmp4 in limb mesenchymes leads to the loss of zeugopod elements and to defective joint articulations (Reversade et al. 2005). During endochondral ossification, condensed cells undergo the differentiation into chondrocytes that secrete an abundance of cartilage matrices including types II, IX, and XI collagen. Sox9 is also the earliest known transcription...