The cohesin complex was first known for roles in sister chromatin cohesion and DNA damage repair. Over the last 15 years, new functions have been described for cohesin in gene expression and genome organization. We previously showed that zebrafish rad21 mutants lack runx1 expression in primitive blood cells. Mutations in the subunits of the cohesin complex, particularly in the STAG2 subunit, have been identified in a range of myeloid malignances. Here, we created isogenic K562 chronic myelogenous leukemia cells with and without the known leukemic STAG2 mutation, R614*. STAG2-/- cells acquired stem cell and extracellular matrix gene expression signatures that were consistent with an observed adherent phenotype. Chromatin accessibility was dramatically altered in STAG2-/- K562, consistent with gene expression changes. Enhanced chromatin accessibility was observed at genes encoding hematopoietic transcription factors, ERG and RUNX1. We show that RUNX1 and ERG transcription is abnormal in STAG2-/- cells following megakaryocyte differentiation with phorbol 12-myristate 13-acetate (PMA). PMA-stimulated STAG2-/- cells showed a striking precocious spike in RUNX1 expression that was associated with enhanced transcription from its proximal P2 promoter. A similar precocious spike was observed in transcription of ERG. Interestingly, the spike in transcription of these genes was confined to early response to stimulation. Treatment of STAG2-/- cells with enhancer blocking BET inhibitor, JQ1, dampened ectopic RUNX1 P2 expression and led to a complete loss of RUNX1 P1 and ERG activation during PMA stimulation. These results suggest that precocious RUNX1 expression in response to STAG2 deficiency is enhancer-driven. Furthermore, JQ1 treatment reduced stem cell-associated c-KIT expression in STAG2-/- mutants. We conclude that STAG2 depletion in leukemic cells amplifies transcription of RUNX1 in response to differentiation signals, and that this characteristic is dampened by BET inhibition. The results have relevance to development of therapeutic strategies for myeloid leukemia.