Exosomes are small extracellular vesicles (sEVs) of ~30-150 nm in diameter that are enriched in exosome marker proteins and play important roles in health and disease. To address large unanswered questions regarding exosome biology in vivo, we created the Exomap1 transgenic mouse, which in response to Cre recombinase expresses the most highly enriched exosomal marker protein known, human CD81, fused to mNeonGreen (HsCD81mNG), and prior to Cre expresses a mitochondrial red fluorescent protein. Validation of the exomap1 mouse with eight distinct Cre drivers demonstrated that HsCD81mNG was expressed only in response to Cre, that murine cells correctly localized HsCD81mNG to the plasma membrane, and that this led to the secretion of HsCD81mNG in EVs that had the size (~70-80 nm), topology, and composition of exosomes. Furthermore, cell type-specific activation of the exomap1 transgene allowed us to use quantitative single molecule localization microscopy to calculate the cell type-specific contribution to biofluid exosome populations. Specifically, we show that neurons contribute ~1% to plasma and cerebrospinal fluid exosome populations whereas hepatocytes contribute ~15% to plasma exosome populations, numbers that reflect the known vascular permeabilities of brain and liver. These observations validate the use of Exomap1 mouse models for in vivo studies of exosome biology.