Ewing's sarcoma is associated with a fusion between the EWS and FLI1 genes, forming an EWS/FLI fusion protein. We developed a system for the identification of cooperative mutations in this tumor through expression of EWS/FLI in primary human fibroblasts. Gene expression profiling demonstrated that this system recapitulates many features of Ewing's sarcoma. EWS/FLI-expressing cells underwent growth arrest, suggesting that growth arrest-abrogating collaborative mutations may be required for tumorigenesis. Expression profiling identified transcriptional upregulation of p53, and the growth arrest was rescued by inhibition of p53. These data support a role for p53 as a tumor suppressor in Ewing's sarcoma and demonstrate the use of transcriptional profiling of model systems in the identification of cooperating mutations in human cancer.