The application of stem cells in drug screening and regenerative therapy has led to important advances in basic biology and biomedicine. Such strategies require high cell numbers and the efficient maturation into faithful functional organ or tissue units. An important aspect to gain control over stem cell fate is the recapitulation of their natural instructive extracellular matrix (ECM). In vivo, stem cells reside in microenvironments, termed niches, providing distinct, tissue-specific signals. In a complex interplay, the fate decisions are tightly regulated by various parameters including biophysical and biochemical signals. Biomaterial science interfaced with sophisticated bioengineering techniques has provided powerful tools to recreate stem cell niches in vitro. However, major challenges for the efficient stem cell expansion and differentiation still have not been sufficiently addressed. In this thesis, platforms have been developed to help to reconstruct this dynamic and niche-specific signaling panoply. In one approach, a hydrogel crosslinking scheme based on a Michael-type addition reaction was combined with photosensitive caging molecules. By masking thiol moieties of a poly(ethylene glycol) (PEG) macromer with photolabile groups, the covalent crosslinking with vinylsulfone groups of complementary PEG macromers was spatiotemporally controlled by light illumination. By the generation of precisely controlled stiffness gradients, we were able to spatially direct human mesenchymal stem cell (hMSC) migration. Along the same lines, light-controlled uncaging was applied to tether biomolecules into a hydrogel matrix. PEG macromers were covalently functionalized with a peptide substrate of activated transglutaminase factor XIII (FXIIIa). This allowed for any desired ECM-derived peptide or protein fused to the counter-reactive peptide substrate to be enzymatically tethered into the hydrogel network. Photoprotection of the hydrogel-bound peptide substrate enabled exquisite dynamic control of biomolecule distribution. The patterning platform was exemplified by the guided three-dimensional (3D) invasion of encapsulated hMSCs into the hydrogel matrix. The second part of this work addressed the challenge of dissecting the vast molecular signaling networks that influence stem cell behavior. A droplet microfluidic-based screening platform was developed to study the effect of pivotal niche proteins in unparalleled combinatorial throughput via micron-sized hydrogel beads termed microniches. Randomized compartmentalization of stable inducible mammalian cell lines into microniches translated into myriads of combinations of secreted proteins tethered to the hydrogel matrix. Selected microniches with a desired phenotypic effect were analyzed for the protein combinations by targeted genotypic analysis of the underlying encapsulated stable mammalian lines. Positive instructive effects of microniches could be demonstrated on a model system of engineered HEK cells and are currently under evaluation for mesendodermal differentiation of a human embryonic stem cell (hESC) reporter cell line. The novel light-responsive materials as well as microfluidic-based screening approach presented here strive for a near-physiological niche signaling architecture. These tools can help to fully exploit the stem cell potential and accelerate the translation of stem cell-based applications from the laboratory towards the clinic.