Extracellular vesicles (EVs) mediate intercellular communication underlying diverse (patho)physiology, including responses to stress, in all organisms studied from bacteria to yeast to human. However, relatively little is understood about the molecular machinery underpinning fundamental EV biology. The Endosomal Sorting Complex Required for Transport (ESCRT) pathway contributes to the biogenesis of small EV populations called exosomes, including selective loading of luminal and membrane protein cargo. Our group discovered that EVs produced by baker’s yeast (Saccharomyces cerevisiae) confer thermotolerance. Herein, my aim is to use this powerful model organism to study functional evolutionary conservation of ESCRT–mediated exosome biogenesis. I replaced yeast VPS23 (yeast vacuolar sorting protein 23), a component of ESCRT-I critical for protein cargo selection, with its human orthologue TSG101, a conventional EV biomarker, tagged it to GFP and evaluated if it confers EV cargo loading, biogenesis and bioactivity. After confirming TSG101-GFP protein expression in yeast by Western blot analysis and fluorescence microscopy, I collected EVs and measured their size by Nanoparticle Tracking Analysis (NTA), total protein content by Bradford assay, and GFP-fluorescence using fluorimetry. EVs were also visualized using transmission electron microscopy to assess structural morphology and confirm size. To assess the capacity to restore bioactivity, I collected EVs from conditioned yeast and measured their ability to provide thermotolerance to naïve cells using a methylene blue- based viability assay and light microscopy. Proteomic analysis by mass spectrometry was conducted to identify EV proteins. In all, I found that human Tsg101-GFP seems to localize at sites of exosome biosynthesis within cells, is present in EVs, and replaces most yeast Vps23 functions in exosome biogenesis, suggesting deep evolutionary conservation of ESCRT–mediated EV biology. These studies set the stage for research focused on better understanding the basis of selective protein cargo loading using S. cerevisiae as a model.