Mesenchymal stromal/stem cells (MSCs) are of great interest in developing new therapies because of their capacity of self-renewal, differentiation and modulation of inflammation or even immunosuppression. However, in vitro expansion is essential before the use of these cells in the clinic, which can lead to changes in the cell properties. The challenge is to optimize the culture conditions and to choose the most appropriate media for the culture of MSC to ensure that they retain their functional properties and thus their therapeutic potential. Moreover, it is necessary to fix parameters related to cell quality that could allow the selection of samples to be used. Currently, the main sources of media supplements used are fetal calf serum (FCS) and human platelet lysate (hPL). The aim of our study was to compare the characteristics and potential of bone marrow and Wharton jelly MSCs cultured in parallel in FCS and in hPL-containing medium. Experiments were performed under hypoxia (2% O2) or normoxia (21% O2) conditions. This study revealed differences in cell behavior depending on the culture condition. The behavior of the cells was related to differential expression of some surface markers and morphological alterations. Cell differentiation capacities were found to be equivalent. These first results confirm the modification of the characteristics of MSCs according to the medium used. These observations must be combined with other functional tests depending on the intended therapeutic application before being able to conclude on the superiority of one culture method over the other.