Mesenchymal Stem Cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS), which is a xenogeneic product and has the potential to alter MSC phenotype, rendering them immunogenic. Thus, efforts have focused on the development of FBS-free media to support the expansion of MSCs. Previous studies have shown that platelet lysate (PL) is an attractive alternative for the expansion of human MSCs. However, there are no detailed long-term MSC functionality studies evaluating the use of equine PL (ePL) for the ex vivo culture of equine MSCs. For our studies, ePL was produced from platelet concentrates following platelet apheresis procedure in equine donors and growth factors concentration was measured. Before suggesting the routine use of ePL for MSC culture, we investigated whether ePL alone could trigger inflammatory responses when exposed to reactive white blood cells such as monocytes. We showed that the addition of ePL to lipopolysaccharide (LPS)-stimulated equine monocytes resulted in significant reduction of pro-inflammatory cytokine production. In our next study, equine bone marrow-derived MSCs in ePL exhibited similar proliferation rates, increased viability, expressed similar levels of MSCs markers and differentiated towards the three lineages, similarly to MSCs grown in FBS. Finally, MSCs cultured in ePL efficiently suppressed the release of pro-inflammatory cytokines when exposed to LPS-stimulated monocytes. Next, we reasoned that the presence of fibrinogen in ePL might be responsible for the less efficient, although not statistically significant, suppression of monocytes activation by MSCs cultured in ePL compared to those in FBS. We removed fibrinogen from ePL (fdePL) and compared the characteristics of MSCs grown in different supplements. The addition of ePL and fdePL on LPS-stimulated monocytes resulted to a statistically significantly decreased cytokine production. MSCs cultured in fdePL media had a statistically significant lower proliferation compared to MSCs in ePL and less pronounced immunomodulatory capacity compared to those cultured in ePL or FBS. In conclusion, ePL suppresses the release of pro-inflammatory cytokines from stimulated monocytes and can be safely used for the expansion of MSCs for clinical purposes. However, fdePL is less efficient at supporting cell proliferation and immunomodulatory capacities of MSCs.