Recent insight into the critical role of pro-inflammatory cytokines, particularly tumor necrosis factor-α (TNF-α), in bone regeneration has heralded a new direction in the design of tissue engineering constructs. Previous studies have demonstrated that continuous delivery of 50 ng/ml TNF-α to mesenchymal stem cells (MSCs) cultured on three-dimensional (3D) biodegradable electrospun poly(ϵ-caprolactone) (PCL) microfiber meshes stimulates mineralized matrix deposition, a marker of osteogenic differentiation. Since TNF-α exhibits a biphasic pattern of expression following bone fracture in vivo, this study aimed to investigate the effects of temporal patterns of TNF-α delivery on in vitro osteogenic differentiation of MSCs cultured on 3D electrospun PCL scaffolds. MSCs were cultured for 16 days and exposed to continuous, early, intermediate, or late TNF-α delivery. To further elucidate the effects of TNF-α on osteogenic differentiation, the study design included MSCs precultured both in the presence and absence of typically required osteogenic supplement dexamethasone. Mineralized matrix deposition was not observed in constructs with dexamethasone-naïve MSCs, suggesting that TNF-α is not sufficient to trigger in vitro osteogenic differentiation of MSCs. For MSCs precultured with dexamethasone, TNF-α suppressed alkaline phosphatase activity, an early marker of osteogenic differentiation, and stimulated mineralized matrix deposition, a late stage marker of MSC osteogenic differentiation. By elucidating the impact of temporal variations in TNF-α delivery on MSC osteogenic differentiation, our results offer insight into the regenerative mechanism of TNF-α and provide the design parameters for a novel tissue engineering strategy that rationally controls TNF-α signaling to stimulate bone regeneration.