The in vitro culture of organoids revolutionized the field of developmental biology, regenerative medicine and preclinical drug discovery. One emerging question is how adult stem cells embedded in Matrigel “spontaneously” undergo morphogenesis? Soluble factors are necessary but not sufficient, suggesting that cues from the matrix are critical. Because Matrigel contains numerous growth factors, it is impossible to parse the role of the matrix from other factors. Here, we took into consideration the mechanical and biological properties of the cell-matrix interaction in vivo, to rationally design fully-defined synthetic matrices that support intestinal stem cell proliferation and differentiation. These matrices can be dissolved on demand to facilitate the recovery of organoids and soluble factors within the hydrogels. PEG macromers are functionalized with either a fibronectin-derived peptide or a collagen-derived peptide via a Michael-type addition reaction. Peptides with affinity to laminin, collagen and fibronectin are also incorporated to capture any cell-secreted polymers. The addition of an MMP-sensitive peptide with or without a sortase-sensitive sequence, allow the recovery of organoids. A partial degradation of the matrix allows organoid morphogenesis. The engineered synthetic matrices support mouse and human intestinal stem cell proliferation form the upper and lower intestinal regions and from inflamed and uninflamed donors. The synthetic matrices are also able to capture donor-to-donor variability to similar extend as Matrigel hydrogels. Organoids emerging in these matrices are enrich in proliferative cells when analyzed by gene expression and respond to basal stimulation to factors such as forskolin, prostaglandin E2 (PGE2) and sodium butyrate, suggesting that these organoids can be used for drug screening studies.