In vivo cellular microenvironments are not well-mimicked in present in vitro cell culture systems. Microtechnology, and microfluidics in particular, provides the tools to create in vivo-like cellular microenvironments in vitro. Features of in vitro cellular microenvironments are discussed and compared to macroscale cell culture environments; the concept of an effective culture volume (ECV) is introduced to facilitate the comparison. Current research using microtechnology to investigate in vitro cellular microenvironments is presented and areas where more research is needed in characterizing the in vitro microenvironment are outlined.