Heterogeneity and ontogeny of mouse thymic macrophages reveal a requirement for Csf1r-expressing myeloid cells during early T cell development

Thymic macrophages (TMs) maintain tissue homeostasis by clearing the large numbers of apoptotic cells generated during T cell development, but how TM heterogeneity relates to their developmental origin and role in thymocyte maturation remains incompletely understood. Using complementary flow-cytometric, single-cell transcriptomic, and genetic approaches, we resolved two major TM populations corresponding to TIMD4 + cortical and CX3CR1 + medullary/cortico-medullary macrophages. TIMD4 + VCAM1 + TMs displayed a prominent efferocytosis and apoptotic-cell-clearance program, whereas TIMD4 - VCAM1 + TMs were enriched for antigen-presentation and interferon-response pathways. Fate mapping revealed unequal progenitor contributions to these populations, and CCR2 deficiency selectively reduced TIMD4 - VCAM1 + TMs, and thymic monocytes, supporting ongoing input from circulating precursors. Exploratory pseudotime analysis further identified a transcriptional continuum from Ly6c2 + Ccr2 + monocytes toward macrophage states. Using MaFIA fetal thymic organ cultures, AP20187-mediated depletion of Csf1r- expressing myeloid cells reduced CD4 + CD8 + thymocyte differentiation and produced a coordinated accumulation of DN3 cells, loss of DN4 cells, and reduction in CD27 expression. These convergent changes identify the DN3-to-DN4 transition as a developmental stage that requires an intact Csf1r -expressing myeloid compartment and establish a functional connection between the thymic myeloid niche and early αβ T cell development. Together, our study refines the phenotypic and developmental organization of mouse TMs and reveals a previously underappreciated requirement for myeloid-cell support during progression through the β-selection checkpoint.