Abstract Cell cycle deregulation and enhanced proliferation of cells is one of the hallmarks of oncogenesis. In Ewing Sarcoma, a highly aggressive pediatric cancer, the chimeric transcription factor EWS-FLI1 deregulates cell cycle by targeting several cell cycle regulators including the E2F family of transcription factors. ChIP-seq studies showed a significant overlap of EWS-FLI1 and E2F3/4 binding in Ewing sarcoma cells. We show that EWS-FLI1 is able to directly activate E2F3, followed by the combinatorial binding of EWS-FLI1 and E2F3 on their target genes synergistically activating their transcription. Furthermore we propose a model where EWS-FLI1 directly exchanges repressive E2F4/p130 by E2F3/pRB thereby driving cells into enhanced cell proliferation. However, so far we were unable to experimentally demonstrate a physical interaction of EWS-FLI1 with any E2F or pocket protein. As an alternative approach to the study of functional synergy between EWS-FLI1 and E2F3, we used time resolved RNA and protein expression data as a basis for mathematical modeling of EWS-FLI1 dependent E2F target gene regulation. By Bayesian model selection, we were able to postulate the formation of a complex between EWS-FLI1 and E2F3 as the most likely explanation for the observed ETS/E2F synergy. This study therefore provides an example of how computational systems approaches can complement experimental data in the discovery of disease mechanisms. This study was supported in part by the Austrian Science Fund (FWF), [grant 22328-B09], and by the 7th framework program of the European Commission, [grant 259348] (‘ASSET’). Citation Format: Raphaela Schwentner, Theodore Papamarkou, Maximilian Kauer, Vassilios Stathopoulos, Fan Yang, Sven Bilke, Paul S. Meltzer, Mark Girolami, Heinrich Kovar. Evidence for E2F/EWS-FLI1 oncoprotein synergism using systems biology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2104. doi:10.1158/1538-7445.AM2015-2104