Stem cell behavior is strongly influenced by the physical and chemical cues originating from the extracellular matrix (ECM). In vivo, ECM signals are displayed in a spatiotemporally complex fashion. Current in vitro models to study the role of ECM signals in regulating stem cell behavior are rather limited in recapitulating this microenvironmental complexity, as they are mostly static and homogeneous. In order to achieve a dynamic control of the physical and biochemical properties of a hydrogel network, we here combined two crosslinking reaction schemes with photochemistry in order to control hydrogel stiffness in space, time and intensity.First, thiol moieties of one of the reactive poly(ethylene glycol) (PEG) macromers undergoing crosslinking into a hydrogel network via Michael-type addition were equipped with caging groups. This prevented their reaction with vinyl sulfone groups on the termini of the complementary PEG macromers. Thus, the crosslinking density of the hydrogel network could be controlled by uncaging with light, directly translating into differential hydrogel stiffness. Using this approach, user-defined two-dimensional stiffness patterns between 3-8 kPa (E modulus) were obtained and shown to influence the directional migration of mesenchymal stem/progenitor cells in a Young?s modulus-dependent manner with constant gradient strength. Cell migration was overall more directed on the photo-patterned substrate compared to the non-patterned region, with a bias towards the stiffer region above 5 kPa.Second, we present an approach based on light-activated enzymatic crosslinking by which the biophysical properties of synthetic hydrogels can be modulated in 3D. A peptide substrate of the activated transglutaminase Factor XIII (FXIIIa), a key ECM crosslinking enzyme, was rendered photosensitive by masking its active site with a photolabile cage group. Covalent incorporation of this caged FXIIIa substrate together with the corresponding acceptor enzyme substrate into PEG hydrogels and subsequent laser-scanning lithography affords highly localized additional crosslinking. By encapsulation of intestinal stem cell-derived organoids followed by the photopatterning of precise three-dimensional stiffness areas we hope to be able to direct their self-organization.These cytocompatible and versatile patterning concepts are highly promising for the manipulation of cell fate in stem cell research, disease modeling, and regenerative medicine.