A complex cell culture environment has been shown to maintain the differentiated state of hepatocytes, yet the mechanisms by which environmental cues selectively maintain liver-specific gene transcription have been unknown. In this paper we show that the hepatic environment regulates the activities of at least three liver-enriched transcription factors, eE-TF, eG-TF/HNF3, and eH-TF, that activate the mouse serum albumin enhancer. eE-TF is a heat-stable factor that has a DNA-binding specificity similar to that of the liver transcription factor C/EBP, but is a distinct protein. eG-TF/HNF3 contributes to the liver-specific transcription of several other serum protein genes. eH-TF binds to a TGTTTGC sequence that occurs at regulatory sites of the albumin promoter, the hepatitis B virus enhancer, and other hepatic genes. eE-TF, eG-TF/HNF3, and eH-TF are regulated by different combinations of the following cell culture conditions: a hormonally defined serum-free medium; an extracellular matrix gel; and a transformation-competent simian virus 40 large T antigen. We propose a regulatory network model to explain how cues from the cell lineage and the extracellular environment coordinately help maintain the activities of transcription factors involved in hepatocyte differentiation.