Glucocorticoids are critical components of combination chemotherapy regimens in paediatric acute lymphoblastic leukemia (ALL). In contrast, glucocorticoids are rarely efficacious in treating myeloid and other non-lymphoid malignancies. Glucocorticoid receptor (GR) binding was shown to be lineage-selective and pre-determined by lineage-specific chromatin landscapes. The binding may further modify chromatin structure to exert biological functions. To determine the mechanism of glucocorticoid resistance associated with chromatin accessibility in ALL, we investigated conserved DNA sequences at lymphocyte-specific open chromatin regions (LSOs). Here, we found PU.1, EBF1 and TCF3, 3 key factors involved in lymphocyte development, were enriched at LSOs associated with RNA regulation and H3K27Ac modification. Performing Chromatin-Immuno Precipitation (ChIP) study in ALL patient-derived xenografts (PDXs), we found that PU.1 bound at an enhancer of a pro-apoptotic gene BIM in a glucocorticoid sensitive PDX ALL-54S before and after dexamethasone treatment, but not in a resistant PDX ALL-50R. TCF3 binding at the BIM enhancer was only enriched after the dexamethasone treatment in ALL-54S rather than ALL-50R. In contrast, EBF1 had no enrichment at the BIM enhancer in both PDXs. PU.1 occupancy has been reported to recruit other factors to modulate DNA methylation. Therefore, we next studied DNA methylation, a key factor that influences chromatin accessibility. We focused on the BIM enhancer and compared its methylation status in PDXs. Glucocorticoid-sensitive ALLs showed significantly lower levels (<25%) of DNA methylation compared to resistant ALLs (40-60%). Interestingly, two myeloid cell lines, K562 and NB4, also showed heavy DNA methylation (67% and 91% respectively). We are now in the progress of defining pioneer factors involved in manipulating chromatin accessibility associated with glucocorticoid resistance in acute myeloid leukemia (AML). These data suggest that lineage-specific pioneer protein PU.1 is critical for regulating chromatin accessibility in ALL cells, which predetermined the lineage-specific actions of the GR. Overall, this study has identified an epigenetic mechanism associated with glucocorticoid resistance in paediatric ALL cells.