Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (iPS-HLCs) are expected to be applicable to large-scale in vitro hepatotoxicity screening systems. Accordingly, methods for generating HLCs from human iPS cells have been improved over the past decade. However, although human hepatocytes have zone-specific characteristics in vivo, there is currently no technique to generate zone-specific HLCs from human iPS cells. Therefore, to generate HLCs with zone-specific properties from human iPS cells, we cultured iPS-HLCs using a parenchymal or nonparenchymal cell-conditioned medium (CM). The results showed that urea production and gluconeogenesis capacity in iPS-HLCs were increased by culturing with cholangiocyte-CM, and glutamine production and drug metabolism capacity in iPS-HLCs were increased by culturing with hepatocyte-CM. It was thus clarified that iPS-HLCs acquire zone 1 hepatocyte-like properties by culturing with cholangiocyte-CM and that iPS-HLCs acquire zone 3 hepatocyte-like properties by culturing with hepatocyte-CM. In addition, we found that WNT inhibitory factor-1 secreted from cholangiocytes, and WNT7B and WNT8B secreted from hepatocytes play important roles in the zone-specific conversion of iPS-HLCs. We hope that our findings will facilitate the application of iPS-HLCs to drug discovery research.