This study defines the chromatin accessibility and transcriptional landscapes in thirteen human primary blood cell types, spanning the hematopoietic hierarchy. The authors developed an optimized ATAC-seq protocol, termed Fast-ATAC, which allows for rapid and high-quality measurements in primary blood cells. They applied this protocol to cells isolated from healthy donors and acute myeloid leukemia (AML) patients, studying a total of 137 samples representing 16 major cell types. The results reveal that distal regulatory elements are more cell type-specific and better capture cell identity compared to mRNA expression levels. Enhancer cytometry, a method to enumerate pure cell types from complex populations, was developed and validated. The study also identifies key regulators of hematopoietic differentiation and reveals the lineage ontogeny of genetic elements linked to human diseases. In AML, chromatin accessibility shows unique regulatory evolution in cancer cells with progressive mutation burden. Single AML cells exhibit mixed regulome profiles of disparate developmental stages, and a method to account for this regulatory heterogeneity identified cancer-specific deviations and implicated HOX factors as key regulators of pre-leukemic HSC characteristics. The findings provide insights into hematopoietic development and disease, highlighting the importance of regulome dynamics in understanding leukemogenesis and disease progression.This study defines the chromatin accessibility and transcriptional landscapes in thirteen human primary blood cell types, spanning the hematopoietic hierarchy. The authors developed an optimized ATAC-seq protocol, termed Fast-ATAC, which allows for rapid and high-quality measurements in primary blood cells. They applied this protocol to cells isolated from healthy donors and acute myeloid leukemia (AML) patients, studying a total of 137 samples representing 16 major cell types. The results reveal that distal regulatory elements are more cell type-specific and better capture cell identity compared to mRNA expression levels. Enhancer cytometry, a method to enumerate pure cell types from complex populations, was developed and validated. The study also identifies key regulators of hematopoietic differentiation and reveals the lineage ontogeny of genetic elements linked to human diseases. In AML, chromatin accessibility shows unique regulatory evolution in cancer cells with progressive mutation burden. Single AML cells exhibit mixed regulome profiles of disparate developmental stages, and a method to account for this regulatory heterogeneity identified cancer-specific deviations and implicated HOX factors as key regulators of pre-leukemic HSC characteristics. The findings provide insights into hematopoietic development and disease, highlighting the importance of regulome dynamics in understanding leukemogenesis and disease progression.