Stem cells play a key role in a wide range of biological processes, in large part due to their ability to self-renew or differentiate into specialized cell types in response to various biological cues. In vivo, stem cells reside in a complex microenvironment, termed niche, that regulates cell fate through intricate combinations of biophysical and biochemical factors. To recapitulate some of these crucial interactions ex vivo and to facilitate a quicker transition to clinical and pharmaceutical applications of stem cells, numerous technologies have been developed in recent years to better control and manipulate the cellular microenvironment. In particular, combining synthetic hydrogels having controllable mechanical and biochemical signaling with microfluidic technologies that can automatically and precisely handle fluids results in unprecedented control over in vitro microenvironmental conditions. In this thesis, novel microfluidic approaches were developed to engineer stem cell niches presenting defined and modular cell-instructive physicochemical cues. In a first approach, a novel platform based on computer-controlled hydrodynamic flow focusing was developed in order to tether steady-state gradients of tagged proteins, using Fc or biotin tags, onto the surface of poly(ethylene glycol) (PEG)-based hydrogels displaying selective capturing proteins (ProteinA or NeutrAvidin). This versatile patterning strategy permitted the generation of complex biomolecule gradients, with fine control over the patterning resolution and shape. Furthermore, the chosen binding schemes enabled parallel and orthogonal gradients of multiple proteins to be formed. As a proof-of-principle, we employed this technology to assess the influence of immobilized leukemia inhibitor factor (LIF) concentration on mouse embryonic stem cell self-renewal. While this technology allowed biomolecule dose effect on stem cell behavior to be investigated, it was limited in its ability to modulate multiple microenvironmental factors simultaneously. To address this limitation, droplet-based microfluidic technology was adopted which allows perturbing microenvironmental conditions in a combinatorial fashion and with nearly unmatched precision and throughput. To this end, microfluidic chips were fabricated for the generation of PEG-based microgels with precisely controlled dimensions and physico-chemical properties. First, we developed a versatile biofunctionalization technique for tethering biomolecules of interest to the reactive PEG microgels. Then, selective peptide- and protein-modified formulations were tested for their ability to promote adhesion and proliferation of various stem cell types in a bioreactor-based suspension culture. Next, programmable modulation of the flows was adopted to generate microgels with varying elasticity, biochemical ligands or both. The different microgel properties were encoded with specific fluorescent markers such that the microenvironment properties could be readily identified via microscopy or flow cytometry. Controlling syringe pumps through computer programming allowed us to generate up to 100 populations of microgels with different elasticities and bioactive ligand concentrations in a single experiment. Thousands of compositionally distinct microgels were then analyzed by the intensity levels of two fluorescent moieties incorporated in the hydrogel network. As a proof-of-principle, we demonstrated that this technology can be adopted to [...]