We have developed a technique for fabricating microfluidic devices from gelatin using a natural crosslinking process. Gelatin, crosslinked with the naturally occurring enzyme transglutaminase is molded to produce microchannels suitable for adherent cell culture and analysis. The autofluorescence of the material was shown to be minimal and within the range of typical background, ensuring utility with analyses using fluorescent dyes and labels would not be affected. Also, normal murine mammary epithelial cells were successfully cultured in the microchannels. The morphology of these adherent epithelial cells was shown to be significantly different for cells grown on rigid tissue culture plastic in either macro- or microscale cultures (even in the presence of a surface coating of gelatin) than those grown on the flexible crosslinked gelatin microchannels. Using these devices, the effects of both the extracellular matrix and soluble factors on cellular behavior and differentiation can be studied in microenvironments that more closely mimic the in vivo environment.