AbstractOsteosarcoma (OS) in human patients is characterized by genetic alteration ofTP53. Osteoprogenitor-specificp53-deleted mice (OSmice) have been widely used to study the process of osteosarcomagenesis. However, the molecular mechanisms responsible for the development of OS upon p53 inactivation remain largely unknown. In this study, we detected prominent RUNX3/Runx3 expression in human and mousep53-deficient OS. Myc was aberrantly upregulated by Runx3 via mR1, a consensus Runx site in theMycpromoter, in a manner dependent onp53deficiency. Reduction of the Myc level by disruption of mR1 or Runx3 knockdown decreased the tumorigenicity ofp53-deficient OS cells and effectively suppressed OS development inOSmice. Furthermore, Runx inhibitors exerted therapeutic effects onOSmice. Together, these results show thatp53deficiency promotes osteosarcomagenesis in human and mouse by allowing Runx3 to induce oncogenic Myc expression.