Ewing sarcoma (EWS) is the second most common malignant bone cancer affecting young children and adolescents. Although most patients achieve long‐lasting remission, patients with metastatic or recurrent disease have very poor outcomes and chemo‐resistance is a significant problem. The majority of Ewing sarcomas are driven by a chromosomal translocation resulting in the EWS‐FLI1 fusion gene. EWS‐FLI1 is an oncogenic transcription factor that plays roles in transcriptional regulation, DNA damage response, cell cycle checkpoint control, and alternative splicing. However, the exact molecular mechanisms behind these functions are not understood fully. Although EWS‐FLI1 is specific to EWS cells and required for tumorigenesis, successfully targeting EWS‐FLI1 has proved challenging. Therefore, other therapeutic targets must be identified to improve outcomes for patients with metastatic or recurrent disease.Using a quantitative PCR array, we identified USP1, a deubiquitinase that functions in both the DNA damage and replication stress responses, as being overexpressed in EWS cell lines compared to its cell of origin, the human mesenchymal stem cell. Importantly, we found USP1 to be essential for EWS cell line proliferation, as growth of these cell lines is arrested with knockdown of USP1. USP1 downregulation has been linked to oncogene‐induced cellular senescence dependent on functional p53. However, because p53 is mutated in EWS, the growth arrest we observe following USP1 downregulation must occur via a p53‐independent mechanism. Therefore, we hypothesize that USP1 overexpression functions to bypass cellular senescence in a p53‐independent manner in response to EWS‐FLI1 induced replication stress, resulting in tumorigenesis. Our studies aim to test this hypothesis in EWS and explore the potential to target USP1 alone or in combination with the current chemotherapy regimen for treating EWS.Support or Funding InformationThis work was supported by Pediatric Cancer pilot funding UNMC and UNMC startup funds to Gargi Ghosal and UNMC Graduate Assistantship Fellowship to Halle Halbkat