Mesenchymal stromal cells (MSCs) are promising therapeutic candidates in a variety of diseases due to having immunomodulatory and pro-regenerative properties. In recent years, MSC-derived small extracellular vesicles (sEVs) have attracted increasing interest as a possible alternative to conventional cell therapy. However, translational processes of sEVs for clinical applications are still impeded by inconsistencies regarding isolation procedures and culture conditions. We systematically compared different methods for sEV isolation from conditioned media of expanded bone marrow-derived MSCs and demonstrated considerable variability of quantity, purity, and characteristics of sEV preparations obtained by these methods. The combination of cross flow filtration with ultracentrifugation for sEV isolation resulted in sEVs with similar properties as compared to isolation by differential centrifugation combined with ultracentrifugation, the latter is still considered as gold standard for sEV isolation. In contrast, sEV isolation by a combination of precipitation with polyethylene glycol and ultracentrifugation as well as cross flow filtration and size exclusion chromatography resulted in sEVs with different characteristics, as shown by surface antigen expression patterns. The MSC culture requires a growth-promoting supplement, such as platelet lysate, which contains sEVs itself. We demonstrated that MSC culture with EV-depleted platelet lysate does not alter MSC characteristics, and conditioned media of such MSC cultures provide sEV preparations enriched for MSC-derived sEVs. The results from the systematic stepwise evaluation of various aspects were combined with culture of MSCs in a hollow fiber bioreactor. This resulted in a strategy using cross flow filtration with subsequent ultracentrifugation for sEV isolation. In conclusion, this workflow provides a semi-automated, efficient, large-scale-applicable, and good manufacturing practice (GMP)-grade approach for the generation of sEVs for clinical use. The use of EV-depleted platelet lysate is an option to further increase the purity of MSC-derived sEVs.