Acute lymphoblastic leukemia (ALL) is associated with significant morbidity and mortality, necessitating further improvements in diagnosis and therapy. Targeted therapies directed against chromatin regulators are emerging as promising approaches in preclinical studies and early clinical trials. Here, we demonstrate an oncogenic role for the protein lysine methyltransferase SETDB2 in leukemia pathogenesis. It is overexpressed in pre-BCR+ ALL and required for their maintenance in vitro and in vivo. SETDB2 expression is maintained as a direct target gene of the chimeric transcription factor E2A-PBX1 in a subset of ALL and suppresses expression of the cell-cycle inhibitor CDKN2C through histone H3K9 tri-methylation, thus establishing an oncogenic pathway subordinate to E2A-PBX1 that silences a major tumor suppressor in ALL. In contrast, SETDB2 was relatively dispensable for normal hematopoietic stem and progenitor cell proliferation. SETDB2 knockdown enhances sensitivity to kinase and chromatin inhibitors, providing a mechanistic rationale for targeting SETDB2 therapeutically in ALL.,Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.