In the intricate landscape of cellular communication, small extracellular vesicles (sEVs) originating from endosomes play crucial roles as mediators and have garnered significant attention in theranostics. Our understanding of sEV biogenesis largely stems from studies on cancer cells, which are vital for diagnostics. However, in therapeutics, where mesenchymal stromal cell (MSC)-derived sEVs are emerging as investigational new drugs, their biogenesis pathways remain largely unexplored. This article explores the parallel narratives of sEV biogenesis in cancer cells and stem cells, specifically using HeLa cells and MSCs as model cell lines. This study investigated the roles of key proteins-hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), signal-transducing adaptor molecule (STAM), tumor susceptibility gene 101 (TSG101), and ALG-2-interacting protein X (ALIX)-as identified in HeLa cells, in the context of MSC-sEV biogenesis. While these proteins show similarities across cell types, a discernible difference arises in their primary functions in regulating sEV biogenesis. The critical role of ALIX in MSC-sEV biogenesis, in particular, underscores its potential as a target for modulating sEVs' yield in regenerative therapies. Through this comparative analysis, we identified shared molecular signatures, offering insights to guide therapeutic interventions and unlock the regenerative potential of stem cells.