DNA methylation is a critical epigenetic regulator in mammalian development. This study presents a whole-genome comparative view of DNA methylation using bisulfite sequencing of three cultured cell types representing progressive stages of differentiation: human embryonic stem cells (hESCs), a fibroblastic derivative of hESCs, and neonatal fibroblasts. The methylation maps were compared with those of mature peripheral blood mononuclear cells (monocytes). Promoter hypomethylation and higher gene body methylation were positively correlated with transcription in all cell types. Exons were more highly methylated than introns, with sharp transitions at exon-intron boundaries, suggesting a role for differential methylation in transcript splicing. Developmental stage was reflected in global methylation levels and non-CpG methylation, with hESCs highest, fibroblasts intermediate, and monocytes lowest. Differentiation-associated differential methylation profiles were observed for developmentally regulated genes, including HOX clusters, homeobox transcription factors, and pluripotency-associated genes. The results highlight the value of high-resolution methylation maps for investigating developmental regulatory mechanisms. DNA methylation is an important epigenetic modification that plays critical roles in cellular differentiation, development, and disease. Hypermethylation is associated with heterochromatin and transcriptional silencing. Correct DNA methylation is critical for X-inactivation, imprinting, and silencing of specific genomic elements. Derangements in DNA methylation patterns are associated with dysregulation of gene expression in cancer cells. In mammals, DNA methylation occurs symmetrically on CpG dinucleotides, although cytosine methylation is not limited to CpG sequences. About 70-80% of CpG sites in mammalian cells are methylated, but their distribution is uneven. CpG dinucleotides are largely concentrated in CpG islands, which are found within the promoters of ~70% of human genes. CpGs in promoter-associated CpG islands tend to be unmethylated, but some are differentially methylated in specific tissues or during development. Most methods used to examine DNA methylation patterns are biased toward CpG-rich regions, using restriction enzymes, affinity enrichment, or bisulfite conversion. These studies have provided a snapshot of methylation status in various cell types but have low resolution and limited genomic coverage. Recent methodological improvements have revealed subtler methylation patterns that correlate with gene expression or cell type. For example, methylation at CpG sites at the edges of promoter-associated CpG islands is inversely correlated with gene expression. Expressed protein-coding genes have low methylation around their promoter regions and high methylation over their gene bodies. Comparison between human pluripotent stem cells and somatic cells revealed cell-type-specific areas of differential methylation. These studies show that as the resolution of the genomic methylation profile increases, new, subtler phenomena are revealedDNA methylation is a critical epigenetic regulator in mammalian development. This study presents a whole-genome comparative view of DNA methylation using bisulfite sequencing of three cultured cell types representing progressive stages of differentiation: human embryonic stem cells (hESCs), a fibroblastic derivative of hESCs, and neonatal fibroblasts. The methylation maps were compared with those of mature peripheral blood mononuclear cells (monocytes). Promoter hypomethylation and higher gene body methylation were positively correlated with transcription in all cell types. Exons were more highly methylated than introns, with sharp transitions at exon-intron boundaries, suggesting a role for differential methylation in transcript splicing. Developmental stage was reflected in global methylation levels and non-CpG methylation, with hESCs highest, fibroblasts intermediate, and monocytes lowest. Differentiation-associated differential methylation profiles were observed for developmentally regulated genes, including HOX clusters, homeobox transcription factors, and pluripotency-associated genes. The results highlight the value of high-resolution methylation maps for investigating developmental regulatory mechanisms. DNA methylation is an important epigenetic modification that plays critical roles in cellular differentiation, development, and disease. Hypermethylation is associated with heterochromatin and transcriptional silencing. Correct DNA methylation is critical for X-inactivation, imprinting, and silencing of specific genomic elements. Derangements in DNA methylation patterns are associated with dysregulation of gene expression in cancer cells. In mammals, DNA methylation occurs symmetrically on CpG dinucleotides, although cytosine methylation is not limited to CpG sequences. About 70-80% of CpG sites in mammalian cells are methylated, but their distribution is uneven. CpG dinucleotides are largely concentrated in CpG islands, which are found within the promoters of ~70% of human genes. CpGs in promoter-associated CpG islands tend to be unmethylated, but some are differentially methylated in specific tissues or during development. Most methods used to examine DNA methylation patterns are biased toward CpG-rich regions, using restriction enzymes, affinity enrichment, or bisulfite conversion. These studies have provided a snapshot of methylation status in various cell types but have low resolution and limited genomic coverage. Recent methodological improvements have revealed subtler methylation patterns that correlate with gene expression or cell type. For example, methylation at CpG sites at the edges of promoter-associated CpG islands is inversely correlated with gene expression. Expressed protein-coding genes have low methylation around their promoter regions and high methylation over their gene bodies. Comparison between human pluripotent stem cells and somatic cells revealed cell-type-specific areas of differential methylation. These studies show that as the resolution of the genomic methylation profile increases, new, subtler phenomena are revealed