Nucleosome positioning (NP) is a fundamental parameter in chromatin packing/unpacking, playing key roles in transcriptional regulation and maintenance of genomic integrity. To better understand NP dynamics in ESCs, we studied two systems: a human embryonic stem cell (hESCs) differentiation system and a mouse embryonic stem cell (mESCs) knockout system. We investigated how NP changes in a hESC differentiation system: WA09 hESCs ISL1+ nascent mesoderm (INM) smooth muscle cells (SMCs), by paired-end sequencing of mononucleosomal DNA fragments generated by micrococcal nuclease (MNase)-digestion (MNase-seq). The analysis reveals that at the promoter and gene body, NP is correlated primarily with transcriptional activity and secondarily with the GC content of the sequence. Pluripotent hESCs also exhibit a more dynamic NP than their differentiated derivatives, indicating that more genes are in the poised state and can be readily activated or silenced once differentiation starts. Surprisingly, the study finds mononucleosomal DNA of hESC to be ~10bp (one helix turn) longer than its differentiated WA09-SMC. Moreover, as fragment length increases, both the GC content and %CpG also increase. Thus, longer nucleosomal DNA, possibly arisen from a larger histone core, could be instrumental in maintaining the remarkable genomic integrity of hESC by reducing mutations such as CT changes.We also studied the NP dynamics in promoters of mESCs and Ext1-/- ESCs, with customized high density Comparative Genomic Hybridization (CGH) arrays. We observed the similar pattern as hESCs that at the promoters, NP is correlated primarily with transcriptional activity and secondarily with the GC content of the sequence. In addition, we observed that genes with decreased expression had corresponding NP change while NP remain unchanged for genes have increased expression in the Ext1-/- ESCs. Interestingly, genes activated in Ext1-/- ESCs have significantly longer mRNA half-life than those silenced on average. Both studies revealed a fundamental level of epigenetic control that was not previously recognized and adds to the complexity of epigenetic mechanisms. In addition, our studies provided a systematic tool of studying global NP with high-throughput approaches including the next generation sequencing and high density CGH arrays.