Programmed cell death-1 (PD-1) belongs to an inhibitory signaling pathway capable of maintaining central and peripheral immune tolerance. Blockage of PD-1 has been identified as a promising immunotherapeutic approach for cancer and chronic infectious diseases. However, it is unknown whether PD-1 pathway regulates stem cell function. It is generally believed that mesenchymal stem cells (MSCs) produce PD-1 ligand, but fail to express PD-1. In this study, we show that neural crest-derived MSCs from dental pulp (MSC-DP), but not MSCs from bone marrow, expressed PD-1. Knocking down PD-1 expression in MSC-DP results in a significantly reduced capacity for cell proliferation and accelerated multipotential differentiation. Mechanistically, we show that PD-1 regulates a SHP2/ERK/Notch cascade to maintain proliferation and a SHP2/ERK/β-catenin cascade to inhibit osteo-/odontogenic differentiation. This study indicates that PD-1 is a key surface molecule controlling cell proliferation and multipotential differentiation of MSC-DP. Through regulating PD-1/SHP2/ERK signaling, we can significantly improve the quality and quantity of culture-expanded MSC-DP for potential clinical therapies.