Cellular differentiation is orchestrated by lineage-specific transcription factors and associated with cell type-specific epigenetic signatures. In the present study, we used stage-specific, epigenetic "fingerprints" to deduce key transcriptional regulators of the human monocytic differentiation process. We globally mapped the distribution of epigenetic enhancer marks (histone H3 lysine 4 monomethylation, histone H3 lysine 27 acetylation, and the histone variant H2AZ), describe general properties of marked regions, and show that cell type-specific epigenetic "fingerprints" are correlated with specific, de novo-derived motif signatures at all of the differentiation stages studied (ie, hematopoietic stem cells, monocytes, and macrophages). We validated the novel, de novo-derived, macrophage-specific enhancer signature, which included ETS, CEBP, bZIP, EGR, E-Box and NF-κB motifs, by ChIP sequencing for a subset of motif corresponding transcription factors (PU.1, C/EBPβ, and EGR2), confirming their association with differentiation-associated epigenetic changes. We describe herein the dynamic enhancer landscape of human macrophage differentiation, highlight the power of genome-wide epigenetic profiling studies to reveal novel functional insights, and provide a unique resource for macrophage biologists.