The supplementation of stem cell culture medium with fetal bovine serum (FBS) for maintenance and propagation of cell lines in vitro is associated with immunogenicity and disease transmission by prions, bacteria, and viruses. Therefore, establishing xenogenic-free cell culture media for the benefit of rational testing conditions for stem cells in terms of eliminating the disadvantages is essential. In parallel, a number of investigations have been carried out on nanoparticle (NP) aided stem cell-based therapies. To use NP-integrated stem cell therapy in clinical applications, NP behavior in xenogenic-free stem cell cultures needs to be understood in detail. Mesoporous silica nanoparticles (MSN) are profusely used in biomedical applications and have also shown great potential in stem cell therapies. One of the strategies to make them compatible with stem cell therapy is to alter their surface functionalization. In line with this notion, the main interest of the present investigation was to study the impact of human serum albumin (HSA) association with differently surface-modified MSN, and rationalize the MSN utilization in xenogenic-free stem cell culture by employing C2C12 myogenic progenitor cells as a model system. Our results revealed that HSA coating on differently surface-modified MSN is a promising strategy to improve the colloidal stability of MSN, stem cell viability, and imparts no adverse effects on the differentiation of stem cells.