Acute myeloid leukemia (AML) is one of the most lethal blood malignancies but in lack of therapies targeting leukemia-specific dependencies. Epigenetic programs are often dysregulated in acute myeloid leukemia (AML) and help enforce an oncogenic state of differentiation arrest. Thus, targeting epigenetic vulnerabilities suggests a new angle of developing AML therapeutics. To identify key epigenetic regulators of AML cell fate, we performed a differentiation-focused CRISPR screen in AML cells. This screen identified the histone acetyltransferase KAT6A as a novel regulator of myeloid differentiation that drives critical leukemogenic gene expression programs. We show that KAT6A is the initiator of a newly-described transcriptional control module in which KAT6A-catalyzed promoter H3K9ac is bound by the acetyllysine reader ENL, which in turn cooperates with a network of chromatin factors to induce transcriptional elongation. Inhibition of KAT6A has strong anti-AML phenotypes in vitro and in vivo, suggesting that KAT6A small molecule inhibitors could be of high therapeutic interest for mono or combinatorial differentiation-based treatment of AML.This work provides new insights of how epigenetic writers and readers cooperate to maintain oncogenic programs. The mechanistic discoveries would also shed light on studies of other types of cancer. Besides significant biological and clinical implications, this work also provides a methodology paradigm for using high-throughput screen to explore cell fate determination in other biological systems.