Ewing sarcoma is an aggressive bone and soft tissue tumor in children and adolescents, with treatment remaining a clinical challenge. This disease is mediated by somatic chromosomal translocations of the EWS gene and a gene encoding an ETS transcription factor, most commonly, FLI1. While direct targeting of aberrant transcription factors remains a pharmacological challenge, identification of dependencies incurred by EWS/FLI1 expression would offer a new therapeutic avenue. We used a combination of super-enhancer profiling, near-whole genome shRNA-based and small-molecule screening to identify cyclin D1 and CDK4 as Ewing sarcoma-selective dependencies. We revealed that super-enhancers mark Ewing sarcoma specific expression signatures and EWS/FLI1 target genes in human Ewing sarcoma cell lines. Particularly, a super-enhancer regulates cyclin D1 and promotes its expression in Ewing sarcoma. We demonstrated that Ewing sarcoma cells require CDK4 and cyclin D1 for survival and anchorage-independent growth. Additionally, pharmacologic inhibition of CDK4 with selective CDK4/6 inhibitors led to cytostasis and cell death of Ewing sarcoma cell lines in vitro and growth delay in an in vivo Ewing sarcoma xenograft model. These results demonstrated a dependency in Ewing sarcoma on CDK4 and cyclin D1 and support exploration of CDK4/6 inhibitors as a therapeutic approach for patients with this disease.