Cell-seeded implants are a regenerative medicine strategy that aims to replace injured tissue and restore tissue function. Pluripotent stem cells are promising cell candidates for the development of regenerative medicine therapies as they have the ability to selfrenew and commit towards numerous cell types. In vivo, stem cells reside in a dynamic niche, a stem cell-specific microenvironment that possesses chemical, biological and mechanical cues, which drive the stem cell fate and renewal. The connection between stem cells and their niche is a two-way relationship consisting of both cell-cell interaction and cell-extracellular matrix (ECM) interactions. An alternative regenerative medicine approach is the manipulation of the stem cell microenvironment. Hence, novel strategies have been developed including 3D biomaterials and bioreactor technologies providing topographical, chemical and mechanical cues to recreate the stem cell niche. Understanding the mechanisms controlling stem cell fate and the dynamic nature of the stem cell niche will enable researchers to replicate this stem cell-specific microenvironment, and therefore, harness and control the valuable attributes which stem cells possess. This chapter elucidates the importance of pluripotent stem cells and their dynamic niche in regenerative medicine. It further presents novel strategies to replicate chemical, topographical and mechanical stimuli which are essential for the regulation of stem cell fate and hence tissue regeneration.