We show that tyrosine phosphorylation of FAK was increased as precartilage condensation occurred, followed by a subsequent decrease in proliferation of in vitro micromass culture of wing bud mesenchymal cells. FAK was associated with fibronectin and paxillin, which were maximal at day 3 of culture. FAK was also associated with signaling molecules such as PLC-gamma and PI3-kinase through c-Src. The beta1 integrin antibody and several inhibitors of signaling molecules such as herbimycin A, U73122, LY294002, as well as cytochalasin D, an actin depolymerizing agent, remarkably decreased tyrosine phosphorylation of FAK and its association with fibronectin and paxillin during condensation. resulting in a marked inhibition of condensation and chondrogenesis. Taken together, our findings suggest that beta1 integrin-mediated interaction of mesenchymal cells and fibronectin signals to accelerate the precartilage condensation through tyrosine phosphorylation of FAK and its association with paxillin. This signaling pathway is required for precartilage condensation and subsequent cartilage nodule formation in chondrogenesis.