A number of solid tumors, such as alveolar rhabdomyosarcoma, synovial sarcoma, and myxoid liposarcoma, are associated with recurrent translocation events that encode fusion proteins. Ewing's sarcoma is a pediatric tumor that serves as a prototype for this tumor class. Ewing's sarcomas usually harbor the (11;22)(q24;q12) translocation. The t(11;22) encodes the EWS/FLI fusion oncoprotein. EWS/FLI functions as an aberrant transcription factor, but the key target genes that are involved in oncogenesis are largely unknown. Although some target genes have been defined, many of these have been identified in heterologous model systems with uncertain relevance to the human disease. To understand the function of EWS/FLI and its targets in a more clinically relevant system, we used retroviral-mediated RNAi to “knock-down” the fusion protein in patient-derived Ewing's sarcoma cell lines. By combining transcriptional profiling data from three of these lines, we identified a conserved transcriptional response to EWS/FLI. The gene that was most reproducibly up-regulated by EWS/FLI was NR0B1. NR0B1 is a developmentally important orphan nuclear receptor with no previously defined role in oncogenesis. We validated NR0B1 as an EWS/FLI-dysregulated gene and confirmed its expression in primary human tumor samples. Functional studies revealed that ongoing NR0B1 expression is required for the transformed phenotype of Ewing's sarcoma. These studies define a new role for NR0B1 in oncogenic transformation and emphasize the utility of analyzing the function of EWS/FLI in Ewing's sarcoma cells. (Mol Cancer Res 2006;4(11):851–7)