Purpose Induced pluripotent stem cells (iPSCs) are promising candidates for regenerative medicine. To overcome the allogeneic immune barrier, we created hypo-immune iPSCs using CRISPR/Cas9 editing strategies and demonstrate long-term survival of allografts without the need of immunosuppression. Methods Hypoimmunogenic mouse and human iPSCs were generated via CRISPR/Cas9 technology by knocking out major histocompatibility complex (MHC) class I and class II and overexpression of the “don't eat me” signal CD47. Unmodified and hypoimmunogenic mouse and human iPSCs were transplanted into allogeneic BALB/c mice or humanized mice, respectively. Cellular and antibody responses as well as innate immune responses were analyzed and in vivo bioluminescence imaging was performed to assess cell survival. Results After allogeneic transplantation, hypoimmunogenic mouse and human cells showed a significantly decreased immune response in ELISPOT assays, comparable to the level of syngeneic transplants. No donor-specific antibodies were detected in mice and humanized mice receiving hypoimmunogenic cells demonstrating a lack of immune recognition. According to the ‘missing-self’ hypothesis, lack of target cell MHC class I expression facilitates NK cell-mediated cytotoxicity. Our data show the existence and functionality of the immune checkpoint signal regulatory protein-α (SIRPα) in innate immune cells which is modulated by CD47 overexpression. The lack of immune activation after transplantation of hypoimmunogenic iPSCs resulted in graft survival of transplanted mouse and human iPSCs in all allogeneic recipients with an observation period of 50 days. Conclusion Hypoimmunogenic iPSCs might serve as an unlimited cell source for the generation of universally-compatible “off-the-shelf” cell grafts or tissues in future clinical applications without the need of immunosuppression.