Primary BM MKs have cellular heterogeneity with 3 functionally distinct subpopulations.
A unique CD53+ MK subpopulation possesses a monocytic-like transcriptional program with immunologic capabilities.
Megakaryocytes (MKs), the platelet progenitor cells, play important roles in hematopoietic stem cell (HSC) maintenance and immunity. However, it is not known whether these diverse programs are executed by a single population or by distinct subsets of cells. Here, we manually isolated primary CD41+ MKs from the bone marrow (BM) of mice and human donors based on ploidy (2N-32N) and performed single-cell RNA sequencing analysis. We found that cellular heterogeneity existed within 3 distinct subpopulations that possess gene signatures related to platelet generation, HSC niche interaction, and inflammatory responses. In situ immunostaining of mouse BM demonstrated that platelet generation and the HSC niche–related MKs were in close physical proximity to blood vessels and HSCs, respectively. Proplatelets, which could give rise to platelets under blood shear forces, were predominantly formed on a platelet generation subset. Remarkably, the inflammatory responses subpopulation, consisting generally of low-ploidy LSP1+ and CD53+ MKs (≤8N), represented ∼5% of total MKs in the BM. These MKs could specifically respond to pathogenic infections in mice. Rapid expansion of this population was accompanied by strong upregulation of a preexisting PU.1- and IRF-8–associated monocytic-like transcriptional program involved in pathogen recognition and clearance as well as antigen presentation. Consistently, isolated primary CD53+ cells were capable of engulfing and digesting bacteria and stimulating T cells in vitro. Together, our findings uncover new molecular, spatial, and functional heterogeneity within MKs in vivo and demonstrate the existence of a specialized MK subpopulation that may act as a new type of immune cell.