Peripheral blood-derived mesenchymal stem cells (PB-MSCs) show promise as a source of cells for autologous transplantation because they can be harvested through minimally invasive procedures. To ensure adequate numbers of cells for transplantation and tissue regeneration, PB-MSCs must first be cultured and expanded in vitro, but whether long-term passage modifies their properties has been poorly understood. In this study we triggered production of PB-MSCs in rabbits using granulocyte colony-stimulating factor (G-CSF) and AMD3100, and then isolated and expanded the cells in culture until they reached a state of senescence, usually after about 20 passages. Cultures of low-, middle-, and high-passage numbers were compared in terms of morphology, proliferative capacity, phenotype, differentiation potential, apoptosis, metabolic indicators, and senescence. As passage number increased, MSCs retained their elongated spindle shape, but became larger and flatter, slowed in growth gradually, and increased proportion of cells showed G1 arrest. The proportions of apoptotic cells, production of reactive oxygen species (ROS), and ADP/ATP ratio increased with passage number. Expression of senescence-associated β-galactosidase increased, while telomerase activity decreased. On the other hand, cultures did not show significant changes in phenotype or lose their ability to differentiate into three lineages as passage number increased. These results suggest that PB-MSCs maintain their stem cell properties during prolonged culturing, but they undergo senescence that may be due to apoptosis and production of ROS. These findings may help to standardize in vitro production of PB-MSCs for tissue engineering.