Human Embryonic Stem Cells (hESC) are unspecialized cells capable of renewing themselves through cell division, and they can be induced to become any tissue- or organ-specific cells with special functions. Epigenetic mechanisms, including DNA methylation, chromatin remodeling and noncoding RNA-mediated processes, have profound regulatory effects in stem cells behavior and fate. Thus, decoding the DNA methylation signature in stem cells genome is vital to understanding the influence of epigenetics in their pluripotency and differentiation. DNA methylation is mainly accomplished by three DNA methyltransferases (DNMTs): The de novo DNMT3A and 3B have the ability to methylate previously unmethylated sequences, whereas the maintenance of DNA methylation during replication relies on DNMT1. DNMT3L (DNMT3-like) lacks enzymatic activity, but is known to be associated to DNMT3A and 3B by modulating their enzymatic activity. Our previous studies comparing human embryonic stem cells (hESCs), their differentiated derivatives, and differentiated primary cells have shown that the level of DNA methylation is inversely correlated with differentiation status. The highest level of methylation was found in the undifferentiated hESCs while the lowest was seen in the fully differentiated fibroblasts. A significant percentage of the DNA methylation in hESCs was observed to be at CpA dinucleotides, rather than canonical CpG dinucleotides. Our current research attempts to establish why non-CpG methylation exists at a significantly higher level in hESCs compared to more differentiated cells and the involvement of de novo DNA methyltransferase 3B on the regulation of both CpG and CpA methylation. In addition we are evaluating the implication of methylation by DNMT3b in hESC differentiation. A better understanding of the role of DNA methylation in regulation of gene expression in pluripotency and differentiation may enable us to more effectively direct cellular differentiation and produce homogeneous cell populations of desired phenotypes for drug development and cell replacement therapies.