ABSTRACT: Human mononuclear phagocytes comprise specialized subsets of dendritic cells (DCs) and monocytes, but how these subsets individually regulate expression of the molecular signals involved in T cell costimulation is incompletely understood. Here, we used multiparameter flow cytometry and CITE-sequencing to investigate the cell type-specific responses of human peripheral blood DC and monocyte subsets to type I interferons (IFN-I), focusing on differential regulation of costimulatory molecules. We report that IFN-β drives the maturation of the recently identified human CD1c⁺ CD5^- DC3 subset into cells with higher GITRL and lower CD86 expression compared with other conventional DC subsets. Transcriptomic analysis confirmed that DC3s have an intermediate phenotype between that of CD1c⁺ CD5⁺ DC2s and CD14⁺ monocytes, characterized by high expression of MHCII, Fc receptors, and components of the phagocyte NADPH oxidase. IFN-β induced a shared core response in human DC and monocyte subsets as well as subset-specific responses, including differential expression of costimulatory molecules. Gene regulatory network analysis suggests that upon IFN-β stimulation NFKB1 drives DC3s to acquire a maturation program shared with DC2s. Accordingly, inhibition of NF-κB activation prevented the acquisition of a mature phenotype by DC3s upon IFN-β exposure. Collectively, this study provides insight into the cell type-specific response of human DC and monocyte subsets to IFN-I and highlights the distinct costimulatory potential of DC3s.