Optimal therapeutic strategies targeting macrophages require in depth understanding of macrophage ontogeny and mechanisms covering their homeostasis. The emerging picture of ontological, functional, and phenotypic heterogeneity within tissue-resident macrophages has altered our understanding of these cells, which play important roles in many human diseases. However, whether the developmental origin has an impact on the functionality of these cells is still unsolved. To answer this question we perform deep immunophenotypic assessment with state of the art multi-parameter flow cytometry and single-cell mass cytometry (Cytometry by Time-Of-Flight mass spectrometry, CyTOF), as well as a combination of in vivo lineage tracking, genetic mouse models and advanced imaging methods.