It has been 20 years since the pioneering work of Shinya Yamanaka and Kazutoshi Takahashi at Kyoto University led to the first successful generation of induced pluripotent stem cells (iPSCs) from mouse embryonic and adult fibroblast cells. iPSCs have the capacity to differentiate into any type of cell in the human body, and as such, they have become ubiquitous in medical and biological research. Throughout the development and use of iPSCs and their derivatives, fluorescent microscopy has been particularly integral, playing a central role in the characterisation of cells and analysis of cellular function of both iPSCs and their derived cells. On the 20th anniversary of their discovery, this short review summarises the development of iPSCs, their application in research and clinical settings, and highlights advances in microscopy and imaging methodologies that have been crucial in developing and characterising iPSCs and their derived cell types.