The development of tissue engineered osteochondral units has been slowed by a number of technical hurdles associated with recapitulating their heterogeneous nature ex vivo. Subsequently, numerous approaches with respect to cell sourcing, scaffolding composition, and culture media formulation have been pursued, which have led to high variability in outcomes and ultimately the lack of a consensus bioprocessing strategy. As such, the objective of this study was to standardize the design process by focusing on differentially supporting formation of cartilaginous and bony matrix by a single cell source in a spatially controlled manner within a single material system. A cell-polymer solution of bovine mesenchymal stem cells and agarose was cast against micromolds of a serpentine network and stacked to produce tissue constructs containing two independent microfluidic networks. Constructs were fluidically connected to two controlled flow loops and supplied with independently tuned differentiation parameters for chondrogenic and osteogenic induction, respectively. Constructs receiving inductive media showed differential gene expression of both chondrogenic and osteogenic markers in opposite directions along the thickness of the construct that was recapitulated at the protein level with respect to collagens I, II, and X. A control group receiving noninductive media showed homogeneous expression of these biomarkers measured in lower concentrations at both the mRNA and protein level. This work represents an important step in the rational design of engineered osteochondral units through establishment of an enabling technology for further optimization of scaffolding formulations and bioprocessing conditions toward the production of commercially viable osteochondral tissue products.