We have developed a dual-chambered bioreactor (DCB) that incorporates a membrane to study stratified 3D cell populations for skin tissue engineering. The DCB provides adjacent flow lines within a common chamber; the inclusion of the membrane regulates flow layering or mixing, which can be exploited to produce layers or gradients of cell populations in the scaffolds. Computational modeling and experimental assays were used to study the transport phenomena within the bioreactor. Molecular transport across the membrane was defined by a balance of convection and diffusion; the symmetry of the system was proven by its bulk convection stability, while the movement of molecules from one flow line to the other is governed by coupled convection-diffusion. This balance allowed the perfusion of two different fluids, with the membrane defining the mixing degree between the two. The bioreactor sustained two adjacent cell populations for 28 days, and was used to induce indirect adipogenic differentiation of mesenchymal stem cells due to molecular cross-talk between the populations. We successfully developed a platform that can study the dermis-hypodermis complex to address limitations in skin tissue engineering. Furthermore, the DCB can be used for other multilayered tissues or the study of communication pathways between cell populations.