In the past several decades, significant efforts have been devoted to recapitulating the in vivo tissue microenvironment within an in vitro platform. However, it is still challenging to recreate de novo tissue with physiologically relevant matrix properties and fluid flow. To this end, this study demonstrates a method to independently tailor matrix stiffness and interstitial fluid flow using a cell-microenvironment-on-a-chip (CMOC) platform. Collagen-polyethylene glycol gels tailored to present controlled stiffness and hydraulic conductivity were fabricated in a microfluidic chip. The chip was assembled to continuously create a steady flow of media through the gel. In the C-MOC platform, interstitial flow mitigated the effects of matrix softness on breast cancer cell behavior, according to an immunostaining-based analysis of estrogen receptor-a (ER-a), integrin and E-cadherin. This advanced cell culture platform serves to engineer tissue similar to in vitro tissue and contribute to better understanding and regulating of the biological roles of extracellular microenvironments.