Simple SummaryLeukemic cells evade chemotherapy due to interactions within the bone marrow microenvironment. We have tested the effect of epigenetic therapy on pediatric AML cells in multicell coculture and in murine models. Epigenetic drug combination followed by chemotherapy was effective in overcoming chemoprotection in multicell coculture and increasing survival inKMT2Arearranged murine models.AbstractAdvances in therapies of pediatric acute myeloid leukemia (AML) have been minimal in recent decades. Although 82% of patients will have an initial remission after intensive therapy, approximately 40% will relapse.KMT2Ais the most common chromosomal translocation in AML and has a poor prognosis resulting in high relapse rates and low chemotherapy efficacy. Novel targeted approaches are needed to increase sensitivity to chemotherapy. Recent studies have shown how interactions within the bone marrow (BM) microenvironment help AML cells evade chemotherapy and contribute to relapse by promoting leukemic blast survival. This study investigates how DNA hypomethylating agent azacitidine and histone deacetylase inhibitor panobinostat synergistically overcome BM niche-induced chemoprotection modulated by stromal, endothelial, and mesenchymal stem cells and the extracellular matrix (ECM). We show that direct contact between AML cells and BM components mediates chemoprotection. We demonstrate that azacitidine and panobinostat synergistically sensitize MV4;11 cells andKMT2Arearranged pediatric patient-derived xenograft lines to cytarabine in multicell coculture. Treatment with the epigenetic drug combination reduced leukemic cell association with multicell monolayer and ECM in vitro and increased mobilization of leukemic cells from the BM in vivo. Finally, we show that pretreatment with the epigenetic drug combination improves the efficacy of chemotherapy in vivo.