Stem cell-based treatments have been suggested as promising candidates to meet the demand for post-stroke therapy targeting cerebral tissue recovery. Recently, mesenchymal stem cells (MSCs) have been discovered to help the reestablishment of blood flow through angiogenesis, paving the way for less invasive and time restricted interventions. Their disadvantage lies in their solely invasive means of isolation from bone marrow biopsy or liposuction, as well as their functional decline, cellular rejection risk and inconsistent quality in large-scale. Recent attention was given to their paracrine actions. Small extracellular vesicles (sEVs) secreted by MSCs show promising potential to serve as non-replicating and non-toxic delivery vehicles. By using sEVs as excretory cargo transportation vehicles to achieve comparable therapeutic effects as their parental MSCs, we investigated their angiogenetic potential in in vitro assays. We preconditioned the cell cultures by MSC exposure to hypoxia (and reoxygenation) and drug-induced acid sphingomyelinase (ASM) inhibition. With our present study we are first to evaluate and clearly display that the pretreatment of MSCs with hypoxia and ASM-inhibition amplifies the pro-angiogenic effects of their secreted sEVs. Furthermore, sEVs obtained from amitriptyline treated MSCs exposed to hypoxia as well as hypoxia and reoxygenation decreased the survival of cerebral microvascular endothelial cells under normoxic experiment conditions but increased endothelial cell viability in hypoxic assay conditions. None of the MSC-sEV preparations induced significant alterations of cerebral microvascular endothelial cell proliferation. In conclusion, our findings highlight the importance of culture conditions for the production and efficacy of MSC-derived sEVs.