We have previously described generation of mesenchymal stem cells (MSCs) from human embryonic and induced pluripotent stem cells. One of the central questions in stem cell biology is to understand how stem cells regulate the decision to self-renew vs. differentiate, at the molecular level. In the current studies we used loss-of-function and gain-of-function analyses in primary human MSCs to demonstrate that BMI1 is a critical regulator for self-renewal and multipotency in this interesting cell type. Knockdown of BMI1 in MSCs reduced self-renewal by upregulation of p16INK4A and increased apoptosis. Knockdown of p16INK4A partially rescued the self-renewal defect in MSCs with loss of BMI1. Overexpressed BMI1 reduced apoptosis and increased cell proliferation by repressing p16INK4A. Loss of BMI1 resulted in deregulation of PPAR, an adipogenic factor, and imprinted gene network (IGN), which blocks osteogenesis. Knockdown of PPAR or IGN in BMI1 defect models restored osteogenesis. Overexpression of BMI1 repressed transcripts of RUNX2 and PPAR, in osteogenesis and adipogenesis, respectively, which lead to decreased lineage specification potential in MSCs. These data show that BMI1 regulates cell proliferation, apoptosis, and differentiation of human MSCs.