The molecular hallmark of the Ewing's family of tumors is the presence of balanced chromosomal translocations, leading to the formation of chimerical transcription factors (that is, EWS/FLI1) that play a pivotal role in the pathogenesis of Ewing's tumors by deregulating gene expression. We have recently demonstrated that DAX1 (NR0B1), an orphan nuclear receptor that was not previously implicated in cancer, is induced by the EWS/FLI1 oncoprotein and is highly expressed in Ewing's tumors, suggesting that DAX1 is a biologically relevant target of EWS/FLI1-mediated oncogenesis. In this study we demonstrate that DAX1 is a direct transcriptional target of the EWS/FLI1 oncoprotein through its binding to a GGAA-rich region in the DAX1 promoter and show that DAX1 is a key player of EWS/FLI1-mediated oncogenesis. DAX1 silencing using an inducible model of RNA interference induces growth arrest in the A673 Ewing's cell line and severely impairs its capability to grow in semisolid medium and form tumors in immunodeficient mice. Gene expression profile analysis demonstrated that about 10% of the genes regulated by EWS/FLI1 in Ewing's cells are DAX1 targets, confirming the importance of DAX1 in Ewing's oncogenesis. Functional genomic analysis, validated by quantitative RT-PCR, showed that genes implicated in cell-cycle progression, such as CDK2, CDC6, MCM10 or SKP2 were similarly regulated by EWS/FLI1 and DAX1. These findings indicate that DAX1 is important in the pathogenesis of the Ewing's family of tumors, identify new functions for DAX1 as a cell-cycle progression regulator and open the possibility to new therapeutic approaches based on DAX1 function interference.Oncogene advance online publication, 30 June 2008; doi:10.1038/onc.2008.203.