Abstract The therapeutic properties of extracellular vesicles (EVs) derived from stem cells and stem-like cells make them promising cell-free alternative to regenerative medicine. However, clinical translation of this technology relies heavily on the ability to manufacture EVs in a scalable, reproducible, cGMP-compliant manner. The choice of cell culture media is a critical component for EV manufacturing. In this study, we used human amniotic epithelial cells (hAECs) as a cell model system to explore the effect of chemically defined serum-free media on EV production. Here we showed that different culture media and different cell culture supplements have variable effects on EV production including culture parameters, EV yield, and EV biogenesis. Cell viability and proliferation rate are not reliable quality indicators of EV manufacturing. The levels of common EV tetraspanins and epitope makers can be impacted by different culture media formulations even when culture parameters are maintained, and EV yield are comparable. This study has uncovered some critical aspects regarding EV production culture media that need to be considered in clinical-grade scalable EV manufacturing.