Abstract The recent demonstration that primary cells from the liver can be expanded in vitro as organoids holds enormous promise for regenerative medicine and disease modeling 1–5 . The use of three-dimensional (3D) cultures based on ill-defined and potentially immunogenic matrices, however, hampers the translation of liver organoid technology into real-life applications 6 . We here used chemically defined hydrogels for the efficient derivation of both mouse and human hepatic organoids. Organoid growth was found to be highly stiffness-sensitive and dependent on yes-associated protein 1 (YAP) activity. However, in contrast to intestinal organoids 7 , YAP-mediated stiffness sensitivity was independent of acto-myosin contractility, requiring instead activation of the Src family of kinases (SFKs). Aberrant matrix stiffness on the other hand led to a shift in the progenitor phenotype, resulting in compromised proliferative capacity. Finally, we demonstrate the unprecedented establishment of biopsy-derived human liver organoids without the use of animal components at any step of the process. Our approach thus opens up exciting perspectives for the establishment of protocols for liver organoid-based regenerative medicine.