DCs develop from multipotent progenitors (MPPs), which commit into DC-restricted common dendritic cell progenitors (CDPs). CDPs further differentiate into classical DCs (cDCs) and plasmacytoid DCs (pDCs). Here, we studied the impact of histone acetylation on DC development in C57BL/6 mice by interfering with histone acetylation and deacetylation, employing histone deacetylase (HDAC) inhibitors. We observed that commitment of MPPs into CDPs was attenuated by HDAC inhibition and that pDC development was specifically blocked. Gene expression profiling revealed that HDAC inhibition prevents establishment of a DC-specific gene expression repertoire. Importantly, protein levels of the core DC transcription factor PU.1 were reduced in HDAC inhibitor-treated cells and consequently PU.1 recruitment at PU.1 target genes Fms-like tyrosine kinase 3 (Flt3), interferon regulatory factor 8 (IRF8), and PU.1 itself was impaired. Thus, our results demonstrate that attenuation of PU.1 expression by HDAC inhibition causes reduced expression of key DC regulators, which results in attenuation of DC development. We propose that chromatin modifiers, such as HDACs, are required for establishing a DC gene network, where Flt3/STAT3 signaling drives PU.1 and IRF8 expression and DC development. Taken together, our study identifies HDACs as critical regulators of DC lineage commitment and development.