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Recreating articular cartilage tri-layered patterning for an engineered in vitro cell construct holds promise for advancing cartilage repair efforts. Our approach involves the development of a mul-tichambered perfusion tissue bioreactor that regulates fluid shear stress levels similar to the gradated hydrodynamic environment in articular cartilage. COMSOL modeling reveals our ta-pered cell chamber design will produce three different shear levels, high in the 22 – 41 mPa range, medium in the 4.5 – 8.4 mPa range, and low in the 2.2 – 3.8 mPa range and distributed across the surface of our mesenchymal stromal cell (MSC) encapsulated construct. In a 14-day bioreactor culture, we assess how fluid shear magnitude and cell vertical location within a 3D construct influence cell chondrogenesis. Notably, Sox9 expression for MSCs cultivated in our reactor shows spatially patterned gene upregulations coding for key chondrogenic marker pro-teins. Beginning with the high shear stress region, lubricin and type II collagen gene increases of 410 and 370-fold indicate cell movement towards a superficial zone architype which is further supported by histological and immunohistochemical stains illustrating the formation of a dense proteoglycan matrix enriched with lubricin, versican, and collagen types I and II molecules. For the medium shear stress region high aggrecan and type II collagen gene expressions of 2.3 and 400-fold, respectively, along with high proteoglycan analyses show movement toward a superfi-cial/mid-zone cartilage architype. For low shear stress regions higher collagen types II and X gene upregulations of 550 and 8,300-fold, the latter being 2x of that for the high shear regime, indicate cell movement with deep zone characteristics. Collectively, biochemical analysis, histol-ogy, and gene expression data demonstrated that our fluid shear bioreactor induced a stratified structure within tissue engineered constructs, demonstrating the feasibility of using this ap-proach to recapitulate the structure of native articular cartilage.