Enthesis between tendon or ligament and bone consists of four layers, tendon/ligament, fibrocartilage, calcified fibrocartilage, and bone. This graded structure gives enthesis strength and protects from rupture. During embryonic development, tendon/ligament, skeletal muscle and cartilage primordia appear independently. Through the subsequent interaction between these primordia, tendon and ligament joints skeletal muscle and cartilage are established as one functional unit. We identified progenitor cells expressing both Scleraxis (Scx) and SRY-box 9 (Sox9), which contributes to the formation of the junctional region between tendon/ligament and cartilage. Scx is a basic helix-loop-helix transcription factor predominantly expressed in tendons and ligaments and is required for maturation of these tissues. Scx deficient mice show hypoplastic formation of tendons and ligaments. Sox9 is an essential transcription factor for chondrogenesis, and mutations in the SOX9 gene causes skeletal dysplasia in human. Scx+/Sox9+ progenitors give rise to chondrocytes and tenocytes or ligamentocytes in and around the attachment sites constructing the prospective enthesis in the axial and the appendicular skeleton. In Scx deficient mice, at embryonic day 13.5, cartilage primordia appeared to be normally formed, but the number of Sox9+ cells decreased in the attachment region of tendons and ligaments, such as patella and deltoid tuberosity of humerus. In the same region, phosphorylation of Smad 1/5 and 3 was also reduced. In addition to defects in tendons and ligaments, we found hypoplastic formation or loss of cartilaginous attachment sites in neonatal Scx deficient mice. At 4 weeks of age, the hierarchal structure observed in control mice was lost at the patella tendon enthesis of Scx deficient mice. Our findings indicate that Scx expression in the Scx+/Sox9+ progenitors is required for establishment of the prospective entheseal region. Taken together with recent reports, I would like to introduce the functional role of Scx in enthesis formation.