Our understanding of Ewing's sarcoma development mediated by the EWS/FLI fusion 
protein has been limited by a lack of knowledge regarding the tumor cell of 
origin. To circumvent this, we analyzed the function of EWS/FLI in Ewing's 
sarcoma itself. By combining retroviral-mediated RNA interference with 
reexpression studies, we show that ongoing EWS/FLI expression is required for 
the tumorigenic phenotype of Ewing's sarcoma. We used this system to define the 
full complement of EWS/FLI-regulated genes in Ewing's sarcoma. Functional 
analysis revealed that NKX2.2 is an EWS/FLI-regulated gene that is necessary for 
oncogenic transformation in this tumor. Thus, we developed a highly validated 
transcriptional profile for the EWS/FLI fusion protein and identified a critical 
target gene in Ewing's sarcoma development.