The mononuclear phagocyte system comprises cells as diverse as monocytes, macrophages, and dendritic cells (DCs), which collectively play key roles in innate immune responses and the triggering of adaptive immunity. Recent studies have highlighted the role of growth and transcription factors in defining developmental pathways and lineage relations within this cellular compartment. However, contributions of miRNAs to the development of mononuclear phagocytes remain largely unknown. In the present study, we report a comprehensive map of miRNA expression profiles for distinct myeloid populations, including BM-resident progenitors, monocytes, and mature splenic DCs. Each of the analyzed cell populations displayed a distinctive miRNA profile, suggesting a role for miRNAs in defining myeloid cell identities. Focusing on DC development, we found miR-142 to be highly expressed in classic FLT3-L–dependent CD4⁺ DCs, whereas reduced expression was observed in closely related CD8α⁺ or CD4⁻CD8α⁻ DCs. Moreover, mice deficient for miR-142 displayed an impairment of CD4⁺ DC homeostasis both in vitro and in vivo. Furthermore, loss of miR-142–dependent CD4⁺ DCs was accompanied by a severe and specific defect in the priming of CD4⁺ T cells. The results of our study establish a novel role for miRNAs in myeloid cell specification and define miR-142 as a pivotal genetic component in the maintenance of CD4⁺ DCs.