Multipotent stem cells (MSCs) are used in various therapeutic applications based on their paracrine secretion activity. Here, we set out to identify and characterize the paracrine factors released during osteoblastogenesis, with a special focus on small non-coding RNAs released in extracellular vesicles (EVs). Bone marrow stem cells (BMSCs) and adipose stem cells (ASCs) from healthy human donors were used as representatives of MSCs. We isolated EVs secreted before and after induction of osteoblastic differentiation and found that the EVs contained a specific subset of microRNAs (miRNAs) and tRNA-derived small RNAs (tsRNA) compared to their parental cells. Osteoblastic differentiation had a larger effect on the small RNA profile of BMSC-EVs relative to ASC-EVs. Our data showed that EVs from different MSC origin exhibited distinct expression profiles of small RNA profiles when undergoing osteoblastogenesis, a factor that should be taken into consideration for stem cell therapy.