The differentiation of embryonic or determined stem cell populations into adult liver fates under known conditions yields cells with some adult‐specific genes but not others, aberrant regulation of one or more genes, and variations in the results from experiment to experiment. We tested the hypothesis that sets of signals produced by freshly isolated, lineage‐dependent mesenchymal cell populations would yield greater efficiency and reproducibility in driving the differentiation of human hepatic stem cells (hHpSCs) into adult liver fates. The subpopulations of liver‐derived mesenchymal cells, purified by immunoselection technologies, included (1) angioblasts, (2) mature endothelia, (3) hepatic stellate cell precursors, (4) mature stellate cells (pericytes), and (5) myofibroblasts. Freshly immunoselected cells of each of these subpopulations were established in primary cultures under wholly defined (serum‐free) conditions that we developed for short‐term cultures and were used as feeders with hHpSCs. Feeders of angioblasts yielded self‐replication, stellate cell precursors caused lineage restriction to hepatoblasts, mature endothelia produced differentiation into hepatocytes, and mature stellate cells and/or myofibroblasts resulted in differentiation into cholangiocytes. Paracrine signals produced by the different feeders were identified by biochemical, immunohistochemical, and quantitative reverse‐transcription polymerase chain reaction analyses, and then those signals were used to replace the feeders in monolayer and three‐dimensional cultures to elicit the desired biological responses from hHpSCs. The defined paracrine signals were proved to be able to yield reproducible responses from hHpSCs and to permit differentiation into fully mature and functional parenchymal cells. Conclusion: Paracrine signals from defined mesenchymal cell populations are important for the regulation of stem cell populations into specific adult fates; this finding is important for basic and clinical research as well as industrial investigations. (HEPATOLOGY 2010;)