Microfluidic gradient systems offer a very precise means to probe the response of cells to graded biomolecular signals in vitro, for example to model how morphogen proteins affect cell fate during developmental processes. However, existing gradient makers are designed for non-physiological plastic or glass cell culture substrates that are often limited in maintaining the phenotype and function of difficult-to-culture mammalian cell types, such as stem cells. To address this bottleneck, we combine hydrogel engineering and microfluidics to generate tethered protein gradients on the surface of biomimetic poly(ethylene glycol) (PEG) hydrogels. Here we used software-assisted hydrodynamic flow focusing for exposing and rapidly capturing tagged proteins to gels in a step-wise fashion, resulting in immobilized gradients of virtually any desired shape and composition. To render our strategy amenable for high-throughput screening of multifactorial artificial cellular microenvironments, a dedicated microfluidic chip was devised for parallelization and multiplexing, yielding arrays of orthogonally overlapping gradients of up to 4 × 4 proteins. To illustrate the power of the platform for stem cell biology, we assessed how gradients of tethered leukemia inhibitory factor (LIF) influence embryonic stem cell (ESC) behavior. ESC responded to LIF gradients in a binary manner, maintaining the pluripotency marker Rex1/Zfp42 and forming self-renewing colonies above a threshold concentration of 85 ng cm−2. Our concept should be broadly applicable to probe how complex signaling microenvironments influence stem cell fate in culture.