Co‐culturing different cell types can be useful to engineer a more in vivo‐like microenvironment for cells in culture. Recent approaches to generating cellular co‐cultures have used microfabrication technologies to regulate the degree of cell–cell contact between different cell types. However, these approaches are often limited to the co‐culture of only two cell types in static cultures. The dynamic aspect of cell–cell interaction, however, is a key regulator of many biological processes such as early development, stem cell differentiation, and tissue regeneration. In this study, we describe a micropatterning technique based on microfabricated multilayer parylene‐C stencils and demonstrate the potential of parylene‐C technology for co‐patterning of proteins and cells with the ability to generate a series of at least five temporally controlled patterned co‐cultures. We generated dynamic co‐cultures of murine embryonic stem cells in culture with various secondary cell types that could be sequentially introduced and removed from the co‐cultures. Our studies suggested that dynamic co‐cultures generated by using parylene‐C stencils may be applicable in studies investigating cellular interactions in controlled microenvironments such as studies of ES cell differentiation, wound healing and development. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2008