BACKGROUND AIMS: Extracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed. METHODS: By combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia). RESULTS: The authors' analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types. CONCLUSIONS: Overall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors' MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling.