Recurring deletions of chromosome 7 and 7q [−7/del(7q)] occur in myelodysplastic syndromes and acute myeloid leukemia (AML) and are associated with poor prognosis. However, the identity of functionally relevant tumor suppressors on 7q remains unclear. Using RNAi and CRISPR/Cas9 approaches, we show that an ∼50% reduction in gene dosage of the mixed lineage leukemia 3 (MLL3) gene, located on 7q36.1, cooperates with other events occurring in −7/del(7q) AMLs to promote leukemogenesis. Mll3 suppression impairs the differentiation of HSPC. Interestingly, Mll3-suppressed leukemias, like human −7/del(7q) AMLs, are refractory to conventional chemotherapy but sensitive to the BET inhibitor JQ1. Thus, our mouse model functionally validates MLL3 as a haploinsufficient 7q tumor suppressor and suggests a therapeutic option for this aggressive disease.