This study evaluated the success rate of expansion, phenotype characterization and differentiation capacity of dental pulp mesenchymal stromal cells (DP-MSCs) in Low glucose Dulbecco`s (DMEM) and Alpha modification of Eagle`s medium (α-MEM). Efficacy of a commercial serum free medium was also evaluated. Dental pulps from extracted third molars of 36 patients was obtained:18 samples immersed in DMEM based media(group DMEM), 18 samples in α-MEM based media (group α-MEM ), 11 of 18 samples of group α-MEM were also cultured in commercial serum free media (group SF). Expansion success rate and surface phenotype was analyzed. Then, osteogenic and adipogenic differentiation were proceeded. Chondrogenesis was induced in group SF and cells with the best expansion rate between the DMEM and α-MEM group. Group α-MEM showed significantly higher expansion success rates than DMEM (X2 test). All groups expressed CD105, CD73, CD90 and CD166, but lacked CD45, CD34 and HLA-DR surface molecules. Group α-MEM expressed significantly higher MSC purity (Wilcoxon Rank Sum Test) and differentiation potential (Fishers Exact Test) than group DMEM. Although group SF had slightly better expansion success rate than DP-MSCs in DMEM, group SF did not surpass MSC purity or differential potential of α-MEM. In this primary study for establishing standardized conditions to expand vast numbers of highquality DP- MSCs, showed that the cells had higher success rate of expansion, level of MSC purity, and differentiation potential in α-MEM based media than DMEM. So, α-MEM is designated as the better in vitro DP-MSC culture medium.