Dendritic cells (DCs) are essential regulators of immune responses; however, transcriptional mechanisms that establish DC lineage commitment are poorly defined. Here, we report that the PU.1 transcription factor induces specific remodeling of the higher-order chromatin structure at the interferon regulatory factor 8 (Irf8) gene to initiate DC fate choice. An Irf8 reporter mouse enabled us to pinpoint an initial progenitor stage at which DCs separate from other myeloid lineages in the bone marrow. In the absence of Irf8, this progenitor undergoes DC-to-neutrophil reprogramming, indicating that DC commitment requires an active, Irf8-dependent escape from alternative myeloid lineage potential. Mechanistically, myeloid Irf8 expression depends on high PU.1 levels, resulting in local chromosomal looping and activation of a lineage- and developmental-stage-specific cis-enhancer. These data delineate PU.1 as a concentration-dependent rheostat of myeloid lineage selection by controlling long-distance contacts between regulatory elements and suggest that specific higher-order chromatin remodeling at the Irf8 gene determines DC differentiation. Display omitted •Irf8 expression defines the earliest dendritic-cell-committed bone marrow progenitor•Irf8 separates the early DC-differentiation program from other myeloid lineages•High PU.1 levels direct higher-order chromatin remodelling to initiate Irf8 expression Dendritic cells (DCs) are essential regulators of immune responses. In this study, Rosenbauer and colleagues report that the PU.1 transcription factor induces specific remodeling of the three-dimensional chromatin structure at the interferon regulatory factor 8 (Irf8) gene to initiate DC fate choice. This report shows that PU.1 acts as a concentration-dependent rheostat of myeloid lineage selection by controlling long-distance contacts between regulatory elements and regulates spatiotemporal Irf8 expression to determine DC differentiation.