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Epigenetic alteration may play a role in age-associated dysfunction of stem cells and predispose to the development of hematological cancers. We analyzed global levels of hematopoietic 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in a cross-sectional study comprising 198 unrelated individuals from four age categories (neonates, 25-30, 70-75, and >90 years old) by liquid chromatography-electrospray ionization-tandem mass spectrometry with multiple reaction monitoring. X-chromosome inactivation (XCI) ratios and telomere length (TL) were measured in all individuals by polymerase chain reaction. Sequencing of epigenetic regulator genes (including TET2, DNMT3A, ASXL1, IDH1, IDH2, and WT1) was performed in the two older subcohorts. We found that global 5hmC levels declined with age in human blood cells (27.5% reduction from birth to old age, p < 0.0005). The levels of 5mC underwent a more modest reduction (2.4% drop) between newborns and the elderly (p < 0.0005). Low 5hmC was associated with increased skewing of XCI (age-adjusted p = 0.0304) and reduced TL (age-adjusted p = 0.0354), both surrogate markers of clonal dominance. Of the 100 individuals over the age of 70, 16 had somatic mutations in TET2, 14 in DNMT3A, and none in IDH1, IDH2, or WT1. Individuals with TET2 mutations had significantly lower 5hmC (relative to unmutated individuals), whereas DNMT3A-mutated subjects did not. However, mutations in TET2 cannot account solely for the decline in 5hmC levels observed with aging because unmutated older individuals also had lower 5hmC levels compared with younger individuals. This suggests that the age-associated decline in 5hmC is multifactorial. Larger prospective studies are needed to determine whether 5hmC reduction is a biomarker of hematological cancer development.