Although stem cells hold tremendous potential for clinical applications, their in vitro manipulation remains very challenging. In vivo, stem cells reside in intricate 3D microenvironments, termed niche, in which many local and systemic extrinsic factors are integrated by the cells to induce controlled fate choices. Niche factors must not only be presented to stem cells in the correct composition, but also in a dynamic and spatially heterogeneous manner to evoke the appropriate response. However, state-of-the-art in vitro culture platforms fail to capture such microenvironmental complexity. In this thesis, a novel hydrogel-based microchip technology was invented to recapitulate in vitro the complexity of native stem cell niches. In this system, stem cells can be exposed to gradients of soluble or extracellular matrix (ECM)-tethered biomolecules. As a first proof of principle, this platform was used to locally influence the behavior of mouse embryonic stem cells (mESC) by exposing them to soluble, matrix-tethered or cell-secreted leukemia inhibitory factor (LIF). mESCs grown in synthetic 3D gels were found to respond to LIF gradients in a spatially regulated manner. In two subsequent studies, the microchip technology was utilized to recapitulate embryonic processes that are characterized by highly dynamic and spatially controlled morphogenetic processes that can currently not be modelled in a 3D context in vitro. First, an in vitro model of the developing central nervous system was conceived. In the developing vertebrate neural tube, dorsoventral identity of neural progenitors is defined by counter gradients of sonic hedgehog (Shh) and bone morphogenetic protein 7 (BMP7). Using the developing neural plate of chick embryos as a model system, a dedicated microchip was developed for exposing the neural progenitors in these tissues to gradients of Shh and BMP7. This approach allowed achieving, for the first time, ex vivo dorsoventral patterning. Secondly, a similar approach was employed towards modeling early human development in vitro. To this end, colonies of human embryonic stem cells (hESC) were exposed to opposing gradients of BMP4 and Noggin which induced mesendoderm differentiation exclusively in specific regions of the multicellular constructs. This study revealed the power of the developed platform to spatially control hESCs self-patterning. In a last set of experiments, the microchip technology was applied to mimic the complex molecular and cellular make-up of the bone marrow niche that regulates the activity of hematopoietic stem cells (HSC). A microchip system was successfully designed to capture the main anatomic features of native bone marrow niches with spatially separated compartments that supported either HSC dormancy or activation. Taken together, this thesis presents a set of innovative microengineering concepts for recapitulating the spatiotemporal complexity of native stem cell microenvironments. The platforms introduced here represent important additions to the currently rather limited set of in vitro tools for the study of complex multicellular phenomena in developmental biology, tissue engineering and regenerative medicine.