The bi-lipid membrane is a defining feature of EVs that differentiate them from other biological complexes or macromolecules. Although the lipid membranes of most if not all EVs are derived from the plasma membranes, EV membranes are unique from plasma membranes. For example, the ratio of sphingomyelin to phosphatidylcholine is twice as high in exosomes as in the corresponding cells. For example the molar ratio of sphingomyelin to phosphatidylcholine in MSC sEVs is 3.3:1 versus 1.6:1 in the cells. EVs are also enriched in rare membrane lipids. For example, exosomes which are small EVs derived from GM1 ganglioside-rich lipid rafts via endosomal biogenesis are also enriched in GM1 gangliosides. Cholera toxin B chain (CTB) which have high binding affinity for GM1 gangliosides can be used to identify and/or isolate exosomes. Two other ligands that bind membrane lipids, namely Annexin V (AV) and Shiga toxin B chain (ST) bind MSC sEVs that are distinct from each other and those that bind CTB. The characteristics of these sEVs and the use of these lipid-binding ligands will be discussed.