Human placenta-derived mesenchymal stem cells (P-MSCs) have drawn increasing attention in the field of stem cell research due to their potential in clinical applications as well as their rich and easy to procure cell source. While studies demonstrating the potential of P-MSCs for therapeutic transplantations have been documented, a clinically compliant procedure for P-MSC expansion in vitro has yet to be established. To this end, previous studies have demonstrated that MSCs of bone marrow and cord blood origins cultured in human cord blood serum (hCBS) are comparable to those cultured in fetal bovine serum (FBS), indicating that hCBS may be an alternative to FBS for the development of in vitro cell expansion procedures free of animal components. However, stem cells from origins other than bone marrow or cord blood, particularly from human placental tissues, which have demonstrated a good potential for clinical applications, have not been characterized under similar conditions. In this study, in an attempt to define a clinically compliant protocol for P-MSC expansion in vitro, we examined the effects of human hCBS as a replacement for FBS on cell proliferation capacity, differentiation potential, MSC-specific phenotypic expression and the genetic stability of P-MSCs in cultures. P-MSCs expanded in vitro in autologous hCBS maintained the capacity of self‑renewal and expressed surface antigens characteristic of bone marrow-derived mesenchymal stem cells. Under differentiation conditions, the P-MSCs expanded in hCBS developed into adipogenic, osteogenic and neurogenic cell phenotypes. Chromosomal karyotyping and single cell gel electrophoresis analysis demonstrated that P-MSCs cultured in autologous hCBS were genetically stable. These results suggest that autologous hCBS may be used as an alternative to FBS for the in vitro expansion of P-MSCs for clinical applications.