Exosomes are small extracellular vesicles (sEVs) of ~30–150 nm in diameter that have the same topology as the cell, are enriched in selected exosome cargo proteins, and play important roles in health and disease. To address large unanswered questions regarding exosome biologyin vivo, we created theexomap1transgenic mouse model. In response to Cre recombinase,exomap1mice express HsCD81mNG, a fusion protein between human CD81, the most highly enriched exosome protein yet described, and the bright green fluorescent protein mNeonGreen. As expected, cell type-specific expression of Cre induced the cell type-specific expression of HsCD81mNG in diverse cell types, correctly localized HsCD81mNG to the plasma membrane, and selectively loaded HsCD81mNG into secreted vesicles that have the size (~80 nm), topology (outside out), and content (presence of mouse exosome markers) of exosomes. Furthermore, mouse cells expressing HsCD81mNG released HsCD81mNG-marked exosomes into blood and other biofluids. Using high-resolution, single-exosome analysis by quantitative single molecule localization microscopy, we show here that that hepatocytes contribute ~15% of the blood exosome population whereas neurons contribute <1% of blood exosomes. These estimates of cell type-specific contributions to blood EV population are consistent with the porosity of liver sinusoidal endothelial cells to particles of ~50–300 nm in diameter, as well as with the impermeability of blood-brain and blood-neuron barriers to particles >5 nm in size. Taken together, these results establish theexomap1mouse as a useful tool forin vivostudies of exosome biology, and for mapping cell type-specific contributions to biofluid exosome populations. In addition, our data confirm that CD81 is a highly-specific marker for exosomes and is not enriched in the larger microvesicle class of EVs.