Allogeneic cell-based therapy is emerging as a promising approach in regenerative medicine. However, rejection of allograft due to mismatch of human leukocyte antigens (HLAs) remains a major concern after transplantation. Here, we generated a homozygous B2M knockout induced pluripotent stem cell (iPSC) line, lacking the expression of HLA class I (HLA-I) molecules, using a CRISPR/Cas9 system. The established iPSC line, MUSIi001-A-1, can serve as an in vitro model for studying immunological responses against allogeneic grafts and provides a prototype for “off-the-shelf” allogeneic cell products for future cell-based therapy.