The unique chromatin signature of ES cells is fundamental to the pluripotency and differentiation of ES cells. One key feature is the poised chromatin state of master developmental genes that are transcriptionally repressed in ES cells but ready to be activated in response to differentiation signals. Poised chromatin in ES cells contains both H3 Lys-4 trimethylation (H3K4me3) and H3 Lys-27 trimethylation (H3K27me3) methylation, indicating activating and repressing potential. However, the contribution of non-covalent chromatin structure to the poised state is not well understood. To address whether remodeling of nucleosomes is important to the poised state, we characterized the function of BAF250a, a key regulatory subunit of the ES cell ATP-dependent Brahma-associated factor (BAF) chromatin remodeling complex (esBAF). Acute deletion of BAF250a disrupted the differentiation potential of ES cells by altering the expression timing of key developmental genes and pluripotent genes. Our genome-wide nucleosome and histone modification analyses indicated that the disruption of gene expression timing was largely due to changes of chromatin structures at poised genes, particularly those key developmental genes mediated by BAF250a. Specifically, BAF250a deletion caused a nucleosome occupancy increase at H3K4me3- and/or H3K27me3-associated promoters. Moreover, H3K27me3 levels and the number of bivalent promoter genes were reduced in BAF250aKOES cells. We revealed that BAF250a ablation led to elevated Brg1 but reduced Suz12 recruitment at nucleosome occupancy-increased regions, indicating an unexpected and complicated role of BAF250a in regulating esBAF and Polycomb repressive complex (PRC) activities. Together, our studies identified that BAF250a mediates esBAF and PRC functions to establish the poised chromatin configuration in ES cells, which is essential for the proper differentiation of ES cells.