This study reports the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing (RRBS) and single-molecule-based sequencing, the authors generated DNA methylation maps covering most CpG islands and a representative sampling of conserved non-coding elements, transposons, and other genomic features in mouse embryonic stem cells, embryonic-stem-cell-derived neural cells, and primary tissues. Key findings include: 1. DNA methylation patterns are better correlated with histone methylation patterns than with genome sequence context. 2. CpG methylation is a dynamic epigenetic mark that undergoes extensive changes during cellular differentiation, particularly in regulatory regions outside core promoters. 3. Analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, reminiscent of patterns observed in some primary tumors. The results establish RRBS as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer, and regenerative medicine. The study also highlights the importance of understanding the kinetics and mechanisms underlying hypermethylation in cultured cells, which may have implications for the accuracy of cellular models generated by in vitro differentiation or manipulation.This study reports the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing (RRBS) and single-molecule-based sequencing, the authors generated DNA methylation maps covering most CpG islands and a representative sampling of conserved non-coding elements, transposons, and other genomic features in mouse embryonic stem cells, embryonic-stem-cell-derived neural cells, and primary tissues. Key findings include: 1. DNA methylation patterns are better correlated with histone methylation patterns than with genome sequence context. 2. CpG methylation is a dynamic epigenetic mark that undergoes extensive changes during cellular differentiation, particularly in regulatory regions outside core promoters. 3. Analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, reminiscent of patterns observed in some primary tumors. The results establish RRBS as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer, and regenerative medicine. The study also highlights the importance of understanding the kinetics and mechanisms underlying hypermethylation in cultured cells, which may have implications for the accuracy of cellular models generated by in vitro differentiation or manipulation.