Surface topography can have significant impact on the cell fate decisions of stem cells. Construction of a biomaterial that can mimic the physiological microenvironment can be used to enhance the maintenance or differentiation of stem cells. Here we manufacture two surfaces with monolayer binary colloidal crystals (BCCs) having different topographies, Si2PM and Si5PM, and examined their effect on maintenance and differentiation of human embryonic stem cells (hESCs). Our results demonstrated that BCCs can be coupled with vitronectin to maintain pluripotency of hESCs during culture, and with a gelatin coating to improve early definitive endoderm differentiation of hESCs using an embryonic body assay (i.e. FOXA2 up-regulation). BCCs can be used as novel cell culture substrates to support stem cell maintenance and differentiation.