With their inherent capability of unlimited self-renewal and unique potential to differentiate into functional cells of the three germ layers, human embryonic stem cells (hESCs) hold great potential in regenerative medicine. A major challenge in the application of hESC-based cell therapy is the allogeneic immune rejection of hESC-derived allografts. We derived dendritic cell-like cells (DCLs) from wild type and CTLA4-Ig/PD-L1 knock-in hESCs, denoted WT DCLs and CP DCLs. The expression of DC-related genes and surface molecules was evaluated, as well as their DCL capacity to stimulate allogeneic T cells and induce regulatory T (Treg) cells in vitro. Using an immune system humanized mouse model, we investigated whether the adoptive transfer of CP DCLs can induce long-term immune tolerance of parental hESC-derived smooth muscle and cardiomyocyte allografts. CP DCLs can maintain immune suppressive properties after robust inflammatory stimulation and induce Treg cells. While CP DCLs survive transiently in vivo, they induce long-term immune tolerance of parental hESC-derived allografts. This strategy does not cause systemic immune suppression but induces immune tolerance specific for DCL-specific HLAs, and thus it presents a safe and effective approach to induce immune tolerance of allografts derived from any clinically approved hESC line. NSFC, leading talents of Guangdong Province Program (No. 00201516), Key R&D Program of Guangdong Province (2019B020235003), Science and Technology Innovation Committee of Shenzhen Municipality (JCYJ20180504170301309), National High-tech R&D Program (863 Program No. 2015AA020310), Shenzhen "Sanming" Project of Medicine (SZSM201602102), Development and Reform Commission of Shenzhen Municipality (S2016004730009), CIRM (DISC2-10559). Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.