Tissue engineering has progressed rapidly due to the availability of numerous technologies pertaining to scaffold's fabrication, cell culture techniques, and an improved understanding of cell-scaffold interactions, both in vitro and in vivo. Recently, biomaterials in the form of nanofibers have been introduced as tissue scaffolds after their successful fabrication by various methods such as electrospinning, self-assembly, and phase separation. The scaffolds made of nanofiber biomaterials have been widely used in various soft- and hard-tissue engineering applications. In this chapter we will focus on the electrospun nanofibrous scaffold because of the promise it holds due its feasibility and flexibility for use in full replication (biomimicking) of natural extracellular matrix (ECM). On the other hand, the discovery of several types of stem cells and their response to electrospun nanofiber biomaterials further help in advancing tissue engineering research. Of particular interest, impact of stem cells behaviors such as isolation, adhesion, proliferation, and differentiation onto the nanofibrous scaffolds will be discussed in the context of tissue engineering and regeneration. Future prospects of nanofiber biomaterials in tissue engineering are also discussed. © 2012 Scrivener Publishing LLC. All rights reserved.