The potential use of human embryonic stem cells (hESCs) in cell-based therapies points out the critical importance of epigenomic evaluation for cell-based therapies. Specifically, DNA methylation appears to be a crucial player in establishing cell fate commitment and lineage choices. In this study, we report the global changes observed on the CpG islands distributed in promoters, gene bodies, and intergenic regions and the major biochemical pathways and genes involved in methylation changes as H9-hESCs turn into a neuronal culture containing medium-sized spiny striatal neurons (MSNs). Using an ontogeny-recapitulating protocol of striatal neuron differentiation, we analyzed DNA methylation profiles during the conversion from pluripotency to neuropotency up to the acquisition of a mature neuronal phenotype. H9-hESCs changed the methylation pattern both through de novo methylation and hypomethylation of specific gene promoters. Bioinformatic analysis allowed us to identify a panel of striatal-associated genes, which were regulated by DNA methylation and differentially expressed during striatal commitment. Importantly, DNA methylation analysis revealed that H9-hESCs did not acquire methylation-based oncogenic properties after differentiation. Indeed, hypermethylation of cancer-associated genes that characterize transformed cells, such as Polycomb repressive complex-associated genes, was not detected in the neuronal cultures. However, the oncosuppressor gene, BCL2L11, became hypermethylated in H9-hESC-derived mature neurons. Whole-genome DNA methylation profiling could become a technological platform to predict the differentiative potential of hESC-derived cultures and establish further biosafety assessment quality control tools of the cell-based products.