Mesenchymal stem/stromal cells (MSCs) are a promising tool for cell-based therapy thanks to their ability to secrete trophic factors and immunomodulatory potential. So far, these cells are used to treat a variety of inflammatory diseases like sepsis or severe graft-versus-host disease (GvHD). Considering the number of cells required for their use in cell therapy (between 1–3 × 106 cells/kg), a primary expansion is necessary. However, after an intravenous injection, few cells are found in tissue lesion or in bone marrow. One hypothesis is that chemotactic signals that guide MSCs to an appropriate environment would be altered during in vitro expansion. It is therefore essential to define better conditions of expansion, particularly with good manufacturing process, to obtain enough MSCs, while preserving their immunomodulatory properties and expression of migration molecules. The aim of this research project is to investigate the effect of cell confluence on migration potential and immunological properties of Wharton’s jelly (WJ) MSCs. This research is investigated with 3 levels confluency, at 50, 80 and 100% of confluence by using good manufacturing practice (GMP). We found a better expression of immunomodulatory molecules (PD-L1) and migratory molecules (CD44, membrane CXCR7) for cells that reached 50% confluence. Furthermore, co-stimulatory molecules appear to be less expressed when MSCs are at low confluency. In conclusion low confluence culture should be privileged to improve immunomodulatory and migration phenotype.