This article highlights the current state of three-dimensional spheroid/tissue formation technologies offering a new experimental platform that is both as reproducible as conventional in vitroexperiments and highly correlated to in vivo conditions. Three-dimensional tissue exhibit higher biological functions and reflect the in vivo context more precisely than classical two-dimensional cultures or monolayers. The applications cover highly efficient drug screening, regenerative medicine and fundamental biological research. Numerous three-dimensional spheroid and tissue formation devices have been developed over the past six decades. In early studies, mass production of spheroids using shaking, and non-adhesive surfaces and scaffolds in flasks was conducted. With the emergence of micro/nano fabrication technology and fundamental understanding of micro/nano fluidics, micro/nano devices capable of forming three-dimensional spheroids in a well-controlled manner have been extensively studied. The cell species composing spheroids include hepatic cells, cancer cells, primary cells and, more recently, stem cells. Formation of hetero-spheroids composed of different cells has also been attempted to further resemble the in vivo conditions. These new trends in spheroid research are particularly highlighted in this review.