The control point by which chondrocytes take the decision between the cartilage differentiation program or the joint formation program is unknown. Here, we have investigated the effect of α5β1 integrin inhibitors and bone morphogenetic protein (BMP) on joint formation. Blocking ofα5β1 integrin by specific antibodies or RGD peptide(arginine-glycine-aspartic acid) induced inhibition of pre-hypertrophic chondrocyte differentiation and ectopic joint formation between proliferating chondrocytes and hypertrophic chondrocytes. Ectopic joint expressed Wnt14,Gdf5, chordin, autotaxin, type I collagen and CD44, while expression of Indian hedgehog and type II collagen was downregulated in cartilage. Expression of these interzone markers confirmed that the new structure is a new joint being formed. In the presence of BMP7, inhibition of α5β1 integrin function still induced the formation of the ectopic joint between proliferating chondrocytes and hypertrophic chondrocytes. By contrast,misexpression of α5β1 integrin resulted in fusion of joints and formation of pre-hypertrophic chondrocytes. These facts indicate that the decision of which cell fate to make pre-joint or pre-hypertrophic is made on the basis of the presence or absence of α5β1 integrin on chondrocytes.