Significance Current challenges in B cell acute lymphoblastic leukemia (B-ALL) include a comprehensive understanding of mechanistic effects of associated oncogenic factors, the development of efficacious therapeutic regimens, and the identification of B-ALL subgroups with characteristic molecular features that can be targeted in cancer treatment. Here we show that MED1 is required for a identified interaction between the Mediator coactivator complex and oncogenic E2A-PBX1, for the proliferation, specifically, of E2A-PBX1–driven leukemic cells and for the activation of E2A-PBX1 target genes. RUNX1 directs the recruitment of E2A-PBX1 to target genes, including cell cycle regulatory E2F5 and survival signaling pre-B cell receptor genes. These results provide insights into mechanisms underlying E2A-PBX1–mediated leukemogenesis and MED1–E2A-PBX1 interactions as potential therapeutic targets.