Hepatitis C virus (HCV) has emerged as a major cause of human liver disease, with ~3% of the world population persistently infected with and more than one million new cases of infection reported annually. In most cases, HCV escapes the immune system and establishes a chronic infection. In the long term, these chronic carriers are at high risk of developing life-threatening liver disease, including cirrhosis and hepatocellular carcinoma. Primary human hepatocytes isolated from patient biopsies represent the most physiologically relevant cell culture model for hepatitis C virus (HCV) infection, but these primary cells are not readily accessible, display individual variability, and are largely refractory to genetic manipulation. Hepatocyte-like cells differentiated from pluripotent stem cells provide an attractive alternative as they not only overcome these shortcomings but can also provide an unlimited source of noncancerous cells for both research and cell therapy. Despite its promise, the permissiveness to HCV infection of differentiated human hepatocyte-like cells (DHHs) has not been explored. We therefore developed a novel infection model based on DHHs derived from human embryonic (hESCs) and induced pluripotent stem cells (iPSCs). DHHs generated in chemically defined media under feeder-free conditions were subjected to infection by both HCV derived in cell culture (HCVcc) and patient-derived virus (HCVser). Pluripotent stem cells and definitive endoderm were not permissive for HCV infection whereas hepatic progenitor cells were persistently infected and secreted infectious particles into culture medium. RNA interference directed toward essential cellular cofactors, such as CyPA and PI4K, in stem cells resulted in HCV-resistant hepatocyte-like cells after differentiation. Interestingly, we also identified a defined transition during the hepatic differentiation process when the cells become permissive for HCV infection. Permissiveness to infection was correlated with induction of the liver-specific microRNA-122 and modulation of cellular factors that affect HCV replication. Further studies using microarray analysis of gene expression profile between non-permissive and permissive cells revealed activation of other putative proviral factors and downregulation of antiviral factors. We then focused on CIDEB, a liver specific gene, whose expression was upregulated during the transition stage. Knocking-down CIDEB by shRNA in hESCs had little effect on the differentiation of the modified cells toward functional hepatocytes, but could inhibit the infection of DHHs by HCVcc. Similar inhibition of HCV infection was also observed when CIDEB was knocked down in Huh-7.5 cells, by the same shRNA and a commercial siRNA. Subsequent detailed studies showed that CIDEB is not required for the steps including viral particle attachment, HCVpp entry, RNA translation and replication, virion assembly and secretion, but involved in the fusion step when viral envelope proteins fuse with endosomal membrane and release the viral RNA into cytosol. CIDEB was also found to be required for infection of Huh-7.5 cells by dengue virus (DENV), through a similar mechanism by facilitating membrane fusion. Surprisingly, upon HCV and DENV particles entry, early endosome markers (Rab5 and EEA1) could be induced to re-distribute to the surface of lipid droplets, colocalizating with CIDEB, further supporting the importance of CIDEB during membrane fusion process. HCV, but not DENV infection could downregulate CIDEB expression in infected cells, through a posttranscriptional manner. Finally, knockout of CIDEB also effectively protected Huh-7.5 cells from being infected by both HCV and DENV. Taken together, the ability to infect cultured cells directly with HCVcc and HCV patient serum, to study defined stages of viral permissiveness, and to produce genetically modified cells with desired phenotypes all have broad significance for host pathogen interactions and cell therapy. Meanwhile, our study also identified a liver-specific HCV entry cofactor that facilitates membrane fusion with a new mechanism and contributes to HCV's hepatic tropism. CIDEB and its interaction with HCV may serve as targets for future anti-HCV therapy.