Human pluripotent stem cells(hPSCs) sense the surrounding chemical, and physical microenvironment and adjust their behaviors accordingly. However, current in vitro research is mostly conducted in plastic culture wares which provides an oversimplified microenvironment compared with in vivo. Recently, increasing evidence has shown that the physical microenvironment plays a critical role in hPSCs differentiation and maintenance. However, current studies were conducted by a bulk culture of hPSCs on substrates with different stiffness. The development of biocompatible materials that allow spatial control of the chemical and mechanical environment for human PSC culture has not yet been achieved. Culture substrates allowing flexible manipulation of physical properties spatially would help us better recapitulate the in vivo environment, and benefit tissue engineering. In this study, we developed a photocurable hydrogel (PVA-PEG gel) which allows us to spatially control the stiffness and the structure at the micrometre level. The generated hydrogel can be functionalized using different ECM proteins. Laminin 511 functionalized PVA-PEG gel supports hPSCs growth and differentiation. Furthermore, by spatially controlling the stiffness of the patterned gel, we could selectively differentiate cells on the same patterned gel, generating complex patterned structures.