Electrospun scaffolds have been studied extensively for their potential use in bone tissue engineering applications. However, inherent issues with the electrospinning approach limit the thickness of these scaffolds and constrain their use for repair of critical-sized bone defects. One method to increase overall scaffold thickness is to bond multiple electrospun scaffolds together with a biocompatible gel. The objective of this study was to determine whether multiple human adipose-derived stem cell (hASC-seeded electrospun, nanofibrous scaffolds could be layered via in situ collagen assembly and whether the addition of laser-ablated micron-sized pores within the electrospun scaffold layers was beneficial to the bonding process. Pores were created by a laser ablation technique. We hypothesized that the addition of micron-sized pores within the electrospun scaffolds would encourage collagen integration between scaffold layers, and promote osteogenic differentiation of hASCs seeded within the layered electrospun scaffolds. To evaluate the benefit of assembled scaffolds with and without engineered pores, hASCs were seeded on individual electrospun scaffolds, hASC-seeded scaffolds were bonded with type I collagen, and the assembled ∼3-mm-thick constructs were cultured for 3 weeks to examine their potential as bone tissue engineering scaffolds. Assembled electrospun scaffolds/collagen gel constructs using electrospun scaffolds with pores resulted in enhanced hASC viability, proliferation, and mineralization of the scaffolds after 3 weeks in vitro compared to constructs using electrospun scaffolds without pores. Scanning electron microscopy and histological examination revealed that the assembled constructs that included laser-ablated electrospun scaffolds were able to maintain a contracted structure and were not delaminated, unlike assembled constructs containing nonablated electrospun scaffolds. This is the first study to show that the introduction of engineered pores in electrospun scaffolds assists with multilayered scaffold integration, resulting in thick constructs potentially suitable for use as scaffolds for bone tissue engineering or repair of critical bone defects.