Abstract Platelet parent cells are megakaryocytes (Mks). Studies have historically focused on Mk derived platelet production in the bone marrow (BM) (1). More recently, platelet-producing Mks have been shown to be present in the lung, and our lab and others have described an immune modulatory phenotype of lung Mks (2-4). The recent identification of a low ploidy, immune differentiated BM Mk sub-population (5) highlights the relationship that lower Mk ploidy may correlate with a more immune differentiated Mk. We refer to the development of a Mk immune modulatory sub-population as Mk immune differentiation. The origin of lung Mks and mechanisms of their immune differentiation remain unknown. It is assumed, without experimental evidence, that lung Mks are ‘seeded’ from immature circulating BM Mks. Lefrancais et al. showed the presence of GFP+ Mks in the lung vasculature when a mTmG lung was transplanted to a PF4-Cre-mTmG recipient. The authors argued that the presence of GFP+ intravascular lung Mks demonstrated an extra-pulmonary source. However, intravascular and extravascular lung Mks have morphological and perhaps functional differences, and extravascular lung Mks make up about 70% of all lung Mks. Thus, the majority of circulating Mks would have to leave the blood and tissue migrate to become lung resident. Without direct evidence for this trafficking, the definitive origin of extravascular lung Mks remains unknown, but may have a major impact on their immune differentiation. I hypothesize that extravascular lung Mks derive from a local hematopoietic progenitor pool independent of BM megakaryopoiesis. I will test this hypothesis by using complementary methods, including irradiation and transfer experiments, lineage tracing, and in vitro colony forming unit assays. The impact of Mk immune differentiation on the platelet pool is still not clear. Lung Mks make platelets, although the relative proportion of lung derived platelets remains controversial (4, 6). Emerging evidence from studies of platelet heterogeneity are beginning to suggest that platelet subsets may have functional significance. I hypothesize that lung resident Mks contribute to an immune-differentiated platelet sub-population. To test this hypothesis, I will use a novel strategy to identify lung derived platelets using oropharyngeal CFSE administration in physiologic and thrombocytopenic conditions such as PF4-Cre-iDTR mice and nonlethal murine malaria. Results from my proposed studies will contribute to an improved understanding of immune differentiated megakaryopoiesis, and its role in coordinated immune responses and platelet immune phenotypes, with direct applications to many vascular inflammatory diseases.