A 3D stem cell culture is an artificially created environment that allows biological cells to grow or interact with surrounding extracellular environment in three dimensions. It can be classified into two types of methods, namely, scaffold techniques and scaffold-free techniques. Solid scaffolds, hydrogels, and other materials are used in scaffold techniques. Recent studies have explored the potentiality of human CD34+ stem cells generated in-vitro agarose gel 3D model to understand the process of bone ossification. Scaffold-free techniques include the use of low adhesion plates, hanging drop plates, micropatterned surfaces, rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting. This book presents recent researches on 3D stem cell culture. Scientists and students actively engaged in the field of stem cell biology will find it full of crucial and unexplored concepts.