Extracellular vesicles (EVs) derived from human mesenchymal stromal cells (hMSC-EVs) have been studied in over 200 preclinical applications and dozens of human clinical trials, underscoring the need for scalable production processes compatible with GMP environments. Most existing 2D and 3D Bioreactor hMSC-EV production processes require a cell expansion stage utilizing undefined components, followed by a wash and medium exchange to remove expansion medium impurities prior to an EV collection phase in a defined medium. Simplifying this 3D process to include cell expansion and EV collection in one medium requires chemically defined growth conditions, a fed-batch medium design, and an efficient process to maximize cell and EV yield, and final product quality. We have developed a chemically defined, scalable fed-batch bioreactor production medium to enable the streamlined and highly efficient production of hMSC-EVs. This study evaluates hMSC-EV production and EV quality across multiple donors and tissues in microcarrier spinner flask cultures using a traditional cell expansion, wash, collect process vs the single-step production process, including scale-up to a 3L stirred tank bioreactor. MSC-EVs were produced from hMSCs (hBM and hUC RoosterVial, 1M) in either RoosterNourish-MSC-XF/RoosterReplenish/RoosterCollect-EV or the new highly productive, chemically defined (HiDef-EV) fed-batch system and collected EVs at set times. HiDef-EV cultures led to increased EV production on days 5, 7, 10 and 12 of culture, while maintaining healthy viable cell profiles. The fed-batch process for hMSC-EV production increased the EV collection window from healthy hMSCs resulting in 2-4x increase in hMSC-EV yield over traditional EV production processes. Elimination of the medium exchange and wash steps resulted in utilization of fewer raw materials, retention rather than disposal of EVs produced during cell growth, and significant reductions in total media used and total cost per billion EVs. Additionally, EV Quality Attributes including size, tetraspanin expression, CD-73 activity, RNA, and lipid content are preserved in the HiDef-EV system. Scale up in 3L Eppendorf bioreactor showed comparable cell growth, EV yields and EV quality between traditional and HiDef-EV process. This highly productive chemically defined EV medium is a simplified, time and cost saving solution for the large-scale production of higher purity hMSC-EVs necessary for extensive clinical investigations.