We studied the expression and function of Runx3 during chondrogenesis and chondrocyte maturation. We found that Runx3 is essential for mediating the early stage of endochondral ossification through cooperation with other Runx family members. Introduction: Runx proteins are spatially and temporally co-expressed during skeletal formation. A cooperative and/or redundant function between these factors was postulated, yet the mechanisms underlying these cooperative effects are unknown. Materials and Methods: Expression patterns of Runx3 transcripts were assessed during mouse embryonic developments and limb bud-derived mesenchymal cell differentiation into mature chondrocytes by real-time RT-PCR. Runx3 protein distribution was also determined by immunohistochemistry in mouse embryos. Runx3 gain and loss of function was performed through overexpression and siRNA knockdown of Runx3 into the limb bud-derived cell line MLB13MYC clone17, respectively. Co-transfection experiments were performed in clone 17 cells using the Runx1 promoter and Runx3 cDNA. Promoter activity was measured by luciferase reporter assay. Results: Both Runx3 isoforms are significantly upregulated at the onset of cartilage mineralization and bone formation in E15.5 mice. This upregulation follows that of Sox9 and is concomitant with that of alkaline phosphatase. Furthermore, Runx3 expression remains high during later stages of embryonic development when the levels of osteocalcin are maximal. We determined the expression patterns of Runx3 during chondrogenesis and chondrocyte maturation using mouse limb bud-derived micromass cultures between days 3 and 21. Whereas Runx3 mRNAs are progressively upregulated between days 3 and 14, it is dramatically downregulated at day 21. Markers of chondrocyte maturation alkaline phosphatase and type X collagen are upregulated and maintained throughout the 21 days of culture. Runx3 role in mediating chondrocyte terminal differentiation through gain and loss of function in MLB13MYC clone17 shows that Runx3 regulates both early and late markers of chondrocyte maturation. Finally, Runx3 transcriptionally inhibits Runx1 expression in chondrocytes. Conclusions: We show a role for Runx3 in mediating stage-specific chondrocyte maturation. Our study clearly suggests that, whereas Runx3 may cooperate with Runx2 to induce chondrocyte terminal differentiation, it inhibits Runx1 expression during late maturation.