The demonstrated ability to differentiate both human embryonic stem cells (hESCs) and patient-derived induced pluripotent stem cells (hiPSCs) into hepatocyte-like cells (HLCs) holds great promise for both regenerative medicine and liver disease research. Here, we determined that, despite an immature phenotype, differentiated HLCs are permissive to hepatitis C virus (HCV) infection and mount an interferon response to HCV infection in vitro. HLCs differentiated from hESCs and hiPSCs could be engrafted in the liver parenchyma of immune-deficient transgenic mice carrying the urokinase-type plasminogen activator gene driven by the major urinary protein promoter. The HLCs were maintained for more than 3 months in the livers of chimeric mice, in which they underwent further maturation and proliferation. These engrafted and expanded human HLCs were permissive to in vivo infection with HCV-positive sera and supported long-term infection of multiple HCV genotypes. Our study demonstrates efficient engraftment and in vivo HCV infection of human stem cell-derived hepatocytes and provides a model to study chronic HCV infection in patient-derived hepatocytes, action of antiviral therapies, and the biology of HCV infection.