Background & Aim With increasing demand for Mesenchymal Stromal Cell (MSC) therapy, there is a need for reproducible chemically defined xeno- and serum-free media for growth of hMSCs to minimize process related variability. In the present study, we compare 4 commercially available xeno- and serum-free media (SFM 1-4) from different manufacturers with traditional Dulbeco's Modified Eagle Media (DMEM) containing fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (hPL). Additionally, SFM from one supplier was further tested in combination with or without hPL. Critical hMSC attributes including cell yield, doubling times (dt), population doublings (pd), morphology, identity through flow cytometry and in vitro potency through the induction of indoleamine 2,3-dioxygenase (IDO) by interferon-gamma (IFN-γ) were recorded. Methods, Results & Conclusion MSC yields were significantly higher in SFM (SFM1: 25600, SFM2: 16000, SFM3: 38267 and SFM4: 21600 cells/cm2) when compared to DMEM containing FBS (2000 cells/cm2) or hPL (4133 cell/cm2). Additionally, cells cultured in all SFMs had spindle shaped morphology and appeared smaller in size (medium diameter in SFM: 17.0 ± 1.3 µm versus hPL: 20.4 µm). Cells grown in SFM conditions had significantly lower doubling times (SFM1: 25.4h, SFM2: 28.8h, SFM3: 27.4h and SFM4: 26.5h vs. FBS: 72.0h and hPL: 47.3h) and higher population doublings (SFM1: 5.7, SFM2: 5.0, SFM3: 6.3 and SFM4: 5.4 versus FBS: 2.0 and hPL: 3.1). This effect was heightened in SFM1 basal media with hPL (yield: 54440 cells/cm2; dt: 20.8h and pd: 6.8) and even further with a combination of SFM1 complete media with fibrinogen depleted hPL (92667 cells/cm2; dt: 18.4h, pd: 7.5). There were no differences in terms of cell identity in different media formulations. All cells were positive (>95% expression) for CD73, CD90, CD105 and negative (<5% expression) for CD14, CD19, CD34, CD45, HLA-DR. Cells cultured in SFM were equally potent as those cultured in hPL or FBS as indicated by enhanced IDO expression when treated with IFN-γ (ΔΔCt; SFM: 15.7 vs FBS: 15.6 and hPL: 16.1). Interestingly, improved IDO expression was observed in cells cultured in SFM1 supplemented with hPL (ΔΔCt: 17.6). In conclusion, hMSCs have markedly faster growth but no impact on identity or in vitro potency when cultured in specific SFM formulations. These markedly enhanced rates of proliferation may provide manufacturing advantages yet the ultimate impact on cell phenotype and product safety is yet to be determined. With increasing demand for Mesenchymal Stromal Cell (MSC) therapy, there is a need for reproducible chemically defined xeno- and serum-free media for growth of hMSCs to minimize process related variability. In the present study, we compare 4 commercially available xeno- and serum-free media (SFM 1-4) from different manufacturers with traditional Dulbeco's Modified Eagle Media (DMEM) containing fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (hPL). Additionally, SFM from one supplier was further tested in combination with or without hPL. Critical hMSC attributes including cell yield, doubling times (dt), population doublings (pd), morphology, identity through flow cytometry and in vitro potency through the induction of indoleamine 2,3-dioxygenase (IDO) by interferon-gamma (IFN-γ) were recorded. MSC yields were significantly higher in SFM (SFM1: 25600, SFM2: 16000, SFM3: 38267 and SFM4: 21600 cells/cm2) when compared to DMEM containing FBS (2000 cells/cm2) or hPL (4133 cell/cm2). Additionally, cells cultured in all SFMs had spindle shaped morphology and appeared smaller in size (medium diameter in SFM: 17.0 ± 1.3 µm versus hPL: 20.4 µm). Cells grown in SFM conditions had significantly lower doubling times (SFM1: 25.4h, SFM2: 28.8h, SFM3: 27.4h and SFM4: 26.5h vs. FBS: 72.0h and hPL: 47.3h) and higher population doublings (SFM1: 5.7, SFM2: 5.0, SFM3: 6.3 and SFM4: 5.4 versus FBS: 2.0 and hPL: 3.1). This effect was heightened in SFM1 basal media with hPL (yield: 54440 cells/cm2; dt: 20.8h and pd: 6.8) and even further with a combination of SFM1 complete media with fibrinogen depleted hPL (92667 cells/cm2; dt: 18.4h, pd: 7.5).