This study demonstrates the engineering of biologically active heart valve leaflets using prenatally available human umbilical cord-derived progenitor cells as the only cell source. Wharton's Jelly-derived cells and umbilical cord blood-derived endothelial progenitor cells were subsequently seeded on biodegradable scaffolds and cultured in a biomimetic system under biochemical or mechanical stimulation or both. Depending on the stimulation, leaflets showed mature layered tissue formation with functional endothelia and extracellular matrix production comparable with that of native tissues. This demonstrates the feasibility of heart valve leaflet fabrication from prenatal umbilical cord-derived progenitor cells as a further step in overcoming the lack of living autologous replacements with growth and regeneration potential for the repair of congenital malformation.