Background-—We aim to generate a line of “universal donor” human induced pluripotent stem cells (hiPSCs) that are nonimmunogenic and, therefore, can be used to derive cell products suitable for allogeneic transplantation. Methods and Results-—hiPSCs carrying knockout mutations for 2 key components (β2 microglobulin and class II major histocompatibility class transactivator) of major histocompatibility complexes I and II (ie, human leukocyte antigen [HLA] I/II knockout hiPSCs) were generated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene-editing system and differentiated into cardiomyocytes. Pluripotency-gene expression and telomerase activity in wild-type (WT) and HLAI/II knockout hiPSCs, cardiomyocyte marker expression in WT and HLAI/II knockout hiPSC-derived cardiomyocytes, and assessments of electrophysiological properties (eg, conduction velocity, action-potential and calcium transient half-decay times, and calcium transient increase times) in spheroid-fusions composed of WT and HLAI/II knockout cardiomyocytes, were similar. However, the rates of T-cell activation before (≈21%) and after (≈24%) exposure to HLAI/II knockout hiPSC-derived cardiomyocytes were nearly indistinguishable and dramatically lower than after exposure to WT hiPSC-derived cardiomyocytes (≈75%), and when WT and HLAI/II knockout hiPSC-derived cardiomyocyte spheroids were cultured with human peripheral blood mononuclear cells, the WT hiPSC-derived cardiomyocyte spheroids were smaller and displayed contractile irregularities. Finally, expression of HLA-E and HLA-F was inhibited in HLAI/II knockout cardiomyocyte spheroids after coculture with human peripheral blood mononuclear cells, although HLA-G was not inhibited; these results are consistent with the essential role of class II major histocompatibility class transactivator in transcriptional activation of the HLA-E and HLA-F genes, but not the HLA-G gene. Expression of HLA-G is known to inhibit natural killer cell recognition and killing of cells that lack other HLAs. Conclusions-—HLAI/II knockout hiPSCs can be differentiated into cardiomyocytes that induce little or no activity in human immune cells and, consequently, are suitable for allogeneic transplantation.