Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the treatment of liver disease and the development of effective in vitro toxicity screening tool. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized differentiation protocol, which produces more than 90% albumin-positive HLCs with no purification process. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating three-dimensional (3D) spheroidal aggregate of HLCs. The 3D differentiation method increased expressions of nuclear receptors that regulate the proper expression of key hepatic enzymes. Furthermore, a significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids and HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated the functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic maturation of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models.