Human hepatoblasts (hHBs) and human hepatic stem cells (hHpSCs) were maintained in serum-free Kubota's medium, a defined medium tailored for hepatic progenitors, and on culture plastic versus hyaluronan hydrogels mixed with specific combinations of extracellular matrix components (e.g., type I collagen and laminin). Nuclear magnetic resonance spectroscopy was used to define metabolomic profiles for each substratum tested. The hHpSCs on culture plastic survived throughout the culture study, whereas hHBs on plastic died within 7-10 days. Both survived and expanded in all hydrogel-matrix combinations tested for more than 4 weeks. Profiles of hundreds of metabolites were narrowed to a detailed analysis of eight, such as glucose, lactate, and glutamine, shown to be significant components of cellular pathways, including the Krebs and urea cycles. The metabolomic profiles indicated that hHpSCs on plastic remained as stem cells expressing low levels of albumin but no alpha-fetoprotein (AFP); those in hydrogels were primarily hHBs, expressing AFP, albumin, and urea. Both hHpSCs and hHBs used energy provided by anaerobic metabolism. Variations in hyaluronan-matrix chemistry resulted in distinct profiles correlating with growth or with differentiative responses. Metabolomic footprinting offers noninvasive and nondestructive assessment of physiological states of stem/progenitor cells ex vivo. Disclosure of potential conflicts of interest is found at the end of this article.