Hepatic stellate cells (HpSTCs) are major regulators of hepatic fibrogenesis in adults. However, their early development in fetal liver is largely unknown. To characterize fetal HpSTCs in the liver, in which hepatic development and hematopoiesis occur in parallel, we determined the phenotypic characteristics of HpSTCs from rat fetal livers, using a strategy focused on vitamin A. Storage of vitamin A in the cytoplasm is a unique characteristic of HpSTCs, permitting identification of them by vitamin A-specific autofluorescence (vA+) when excited with UV light using flow cytometry. A characteristic vA+ cell population was identified in liver as early as 13 days post coitum; it had a surface phenotype of RT1A- intercellular adhesion molecule (ICAM)-1+ vascular cell adhesion molecule (VCAM)-1+ beta3-integrin+. Although nonspecific autofluorescent cells were found with the antigenic profile of RT1A- ICAM-1+ VCAM-1+, they were beta3-integrin- and proved to be hepatoblasts, bipotent hepatic parenchymal progenitors. In addition to expression of classic HpSTC markers, the vA+ cells were able to proliferate continuously in a serum-free hormonally defined medium containing leukemia inhibitory factor, which was found to be a key factor for their replication. These results demonstrated that the vA+ cells are fetal HpSTCs with extensive proliferative activity. Furthermore, the vA+ cells strongly express hepatocyte growth factor, stromal-derived factor-1alpha, and Hlx (homeobox transcription factor), indicating that they play important roles for hepatic development and hematopoiesis. The abilities to isolate and expand fetal HpSTCs enable further investigation into their roles in early liver development and facilitate identification of possibly novel signals of potential relevance for liver diseases.