Current stem cell therapy mainly targets diseases that can be treated by cell transplantation. The complexity of organogenesis hinders in vitro generation of organs derived from patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. To address this issue, we attempted in vivo generation of PSC-derived pancreas using Pdx1(-/-) blastocysts(pancreatogenesis-disabled) and blastocyst complementation technique. When wild type rat PSCs were injected into mouse Pdx1(-/-) blastocysts, defective cells were totally replaced and pancreas was formed almost entirely by injected rat PSC derived cells. Chimeric mice of Pdx1(-/-) genotype survived to adulthood without any sign of diabetes. Generation of organs using blastocyst complementation in vivo provides a new strategy for understanding organogenesis and a novel approach for organ supply.