Coordinated activities of multiple mesenchymal cell types contribute to the development of the mammalian skeleton formed through endochondral ossification. Synthesis of a cartilage template by chondrocytes is an obligatory step for the generation of skeletal elements during endochondral ossification. Gene ablation studies have established that Runx2 is an essential transcription factor for bone formation and the differentiation of skeletal cells. However, global gene deletion has failed to discern the tissue- and cell type-specific roles of Runx2. We generated floxed mice to elucidate the Runx2 regulatory control distinctive to cartilage tissue during bone development. Exon 8 of the Runx2 gene was selectively deleted in developing chondrocytes by utilizing Col2a-Cre mice. Cell- and tissue-specific gene recombination was confirmed by β-gal activity in R26R mice. The chondrocyte-specific loss of Runx2 caused failure of endochondral ossification, impaired craniofacial development, dwarfism, and perinatal lethality. Radiographic imaging and histochemical approaches were used to characterize the skeletal phenotype. We conclude that regulatory control of Runx2 in chondrocytes is essential for endochondral ossification, and it is independent of the role of Runx2 in osteoblasts. Copyright © 2011 S. Karger AG, Basel.