Developing physiologically relevant in vitro models for studying periodontitis is crucial for understanding its pathogenesis and developing effective therapeutic strategies. In this study, we aimed to integrate the spheroid culture of periodontal ligament stem cells (PDLSCs) within a spheroid-on-chip microfluidic perfusion platform and to investigate the influence of interstitial fluid flow on morphogenesis, cellular viability, and osteogenic differentiation of PDLSC spheroids. PDLSC spheroids were seeded onto the spheroid-on-chip microfluidic device and cultured under static and flow conditions. Computational analysis demonstrated the translation of fluid flow rates of 1.2 μl min-1 (low-flow) and 7.2 μl min-1 (high-flow) to maximum fluid shear stress of 59 μPa and 360 μPa for low and high-flow conditions, respectively. The spheroid-on-chip microfluidic perfusion platform allowed for modulation of flow conditions leading to larger PDLSC spheroids with improved cellular viability under flow compared to static conditions. Modulation of fluid flow enhanced the osteodifferentiation potential of PDLSC spheroids, demonstrated by significantly enhanced alizarin red staining and alkaline phosphatase expression. Additionally, flow conditions, especially high-flow conditions, exhibited extensive calcium staining across both peripheral and central regions of the spheroids, in contrast to the predominantly peripheral staining observed under static conditions. These findings highlight the importance of fluid flow in shaping the morphological and functional properties of PDLSC spheroids. This work paves the way for future investigations exploring the interactions between PDLSC spheroids, microbial pathogens, and biomaterials within a controlled fluidic environment, offering insights for the development of innovative periodontal therapies, tissue engineering strategies, and regenerative approaches.