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Liver-on-a-Chip technology holds considerable potential for applications in drug screening and chemical-safety testing. To establish such platforms, functional hepatocytes are required; however, primary hepatocytes are commonly used, despite problems involving donor limitations, lot-to-lot variation, and unsatisfactory two-dimensional culture methods. Although human pluripotent stem cells (hPSCs) may represent a strong alternative contender to address the aforementioned issues, remaining technological challenges include the robust, highly efficient production of high-purity hepatic clusters. In addition, current Liver-on-a-Chip platforms are relatively complicated and not applicable for high-throughput experiments. Here, we develop a very simple Liver-on-a-Chip platform with mature and functional hepatocyte-like cells derived from hPSCs. To establish a method for hepatic differentiation of hPSCs, cells were first treated by inhibiting the phosphoinositide 3-kinase- and Rho-associated protein kinase-signaling pathways to stop self-renewal and improve survival, respectively, which enabled the formation of a well-defined endoderm and facilitated hepatocyte commitment. Next, a simple microfluidic device was used to create a three-dimensional (3D) culture environment that enhanced the maturation and function of hepatocyte-like cells by increasing the expression of both hepatic maturation markers and cytochrome P450. Finally, we confirmed improvements in hepatic functions, such as drug uptake/excretion capabilities, in >90% of 3D-matured hepatocyte-like cells by indocyanin green assay. These results indicated that the incorporation of hPSC-derived hepatocytes on our Liver-on-a-Chip platform may serve to enhance the processes involved in drug screening and chemical-safety testing.