During chronic liver injury, inflammation leads to liver fibrosis— particularly due to the activation of hepatic stellate cells (HSCs). However, the involvement of inflammatory cytokines in HSC activation and the relationship between each type of liver cells is still unclear. To examine their interactions, many existing in vitro liver models were performed in 3D organoid or spheroid culture with random 3D structure, which complicates analysis. Herein, we demonstrated the hierarchical coculture of primary rat hepatocytes with non-parenchymal cells such as the human-derived HSC line (LX-2) and the human-derived liver sinusoidal endothelial cell line (TMNK-1). The cocultured tissue had high usability with simple operation of separating solid and liquid phases with improved liver functions such as albumin production and hepatic cytochrome P450 3A4 activity. We also studied the effects of stimulation by both oxygen tension and the key pro-fibrogenic cytokine, transforming growth factor beta (TGF-β), on HSC activation. Gene expression analysis revealed that lower oxygen tension and TGF-β1 stimulation enhanced collagen type I and alpha-smooth muscle actin expression from LX-2 cells in the hierarchical coculture after TGF-β1 stimulation. Therefore, this hierarchical in vitro cocultured liver tissue could provide an improved platform as a disease model for elucidating the interactions of various liver cell types and biochemical signals in future liver fibrogenesis studies.