AbstractTo define the functional role of Krüppel‐like factor (KLF) 10 as a modulator of chondrocyte hypertrophy in developing skeleton, the developmental features in the long bone of KLF10 knockout (KO) mice were investigated and the mesenchymal stem cells (MSCs) from KLF10 KO mice were characterized regarding chondrogenesis and osteogenesis. Delayed long bone growth and delayed formation of primary ossification center were observed in an early embryonic stage in KLF10 KO mouse along with very low Indian hedgehog expression in epiphyseal plate. While the chondrogenic potential of mouse MSCs from KLF10 KO mice appeared normal or slight decreased, hypertrophy and osteogenesis were extensively suppressed. These findings suggest that KLF10 is a mediator of chondrocyte hypertrophy in developing skeleton, and that suppression of KLF10 may be employed as a new strategy for preventing hypertrophy in cartilage regeneration using MSCs.