Dysregulated transcription is a hallmark of cancer cells. However, the reliance of cancer to neoplastic gene signatures established by cancer-specific transcription factors exposes potential therapeutic vulnerabilities. Sequential targeting of transcription factors is a powerful strategy where small molecules inhibit distinct steps along the transcriptional cascade to break down established neoplastic gene signatures. We apply this sequential targeting strategy to Ewing sarcoma, a malignant bone and soft tissue tumor that depends on a unique oncogenic transcription factor, EWS/FLI, to drive disease progression and cancer cell survival. We demonstrate that the DNA binding agent mithramycin decreases the transcription driven by EWS/FLI by disrupting initiation and the accessibility of enhancer regions. We next demonstrate that this effect is greatly augmented with CDK9 inhibition which limits the ability of RNA polymerase II to promote active elongation along mRNA transcripts. This synergy can be accomplished with a combination therapy of mithramycin and the CDK9 inhibitor PHA-767491. We further show that an extended exposure to mithramycin leads to CDK9 inhibition in addition to its anti-EWS/FLI effects leading to a synergistic repression of the EWS/FLI gene signature. Both the combination therapy and the extended exposure schedule lead to striking responses in cell lines and animal models.