Fetal liver cell fractions are potent sources of cells for future liver tissue engineering, by virtue of their high proliferation capacity and their potential for hepatic maturation. Recently, some types of hepatic differentiation agents have been identified from findings in stem cell biology. We therefore investigated the in vitro growth and maturation of rat fetal liver cells isolated from 17-day-old pregnant rats in poly-L-lactic acid three-dimensional (3D) macroporous scaffolds in the presence of soluble factors, such as a combination of hepatocyte growth factor, fibroblast growth factor-1, and fibroblast growth factor-4, oncostatin M, and sodium butyrate. Inclusion of all these factors in the 3D culture induced higher levels of hepatic functions and well maintained these enhanced levels during 2 weeks of culture, whereas in the monolayer culture, such functional enhancement was gradually lost after 1 week. The finally achieved functions on a per-cell basis in the 3D culture with all of the soluble factors were comparable to those of adult hepatocytes. We therefore conclude that the 3D culture system shows promise for the in vitro maturation of fetal liver cells as a means of preconditioning of the cells for engineered liver tissue equivalents in future transplantation studies.