Tissue engineering focuses on the repair and regeneration of tissues through the use of biodegradable scaffold systems that structurally support regions of injury while recruiting and/or stimulating cell populations to rebuild the target tissue. Within bone tissue engineering, the effects of scaffold architecture on cellular response have not been conclusively characterized in a controlled-density environment. We present a theoretical and practical assessment of the effects of polycaprolactone (PCL) scaffold architectural modifications on mechanical and flow characteristics as well as MC3T3-E1 preosteoblast cellular response in an in vitro static plate and custom-designed perfusion bioreactor model. Four scaffold architectures were contrasted, which varied in inter-layer lay-down angle and offset between layers, while maintaining a structural porosity of 60 ± 5%. We established that as layer angle was decreased (90° vs. 60°) and offset was introduced (0 vs. 0.5 between layers), structural stiffness, yield stress, strength, pore size, and permeability decreased, while computational fluid dynamics-modeled wall shear stress was increased. Most significant effects were noted with layer offset. Seeding efficiencies in static culture were also dramatically increased due to offset (∼45% to ∼86%), with static culture exhibiting a much higher seeding efficiency than perfusion culture. Scaffold architecture had minimal effect on cell response in static culture. However, architecture influenced osteogenic differentiation in perfusion culture, likely by modifying the microfluidic environment. Biotechnol. Bioeng. 2014;111: 1440–1451. © 2014 Wiley Periodicals, Inc.