The neural crest (NC) is an attractive system for investigating the mechanisms underlying cell lineage diversification in higher vertebrates. The NC contains a mixed population of already defined precursors and multipotent cells that can give rise to a great variety of cell types, including glial cells and neurons of the peripheral nervous system, melanocytes, and smooth muscle cells (SMCs). Microenvironmental factors, such as the fibroblast growth factor 2 (FGF2), found along migratory paths and in target tissues, strongly influence the fate of multipotent NC precursors. We have previously demonstrated that the FGF2 promotes the differentiation of NC cells to glial phenotypes, while the epidermal growth factor induces NC differentiation to neurons and melanocytes. In the present study, we used mass cultures and single-cell culture assays to demonstrate that FGF2 influences NC cell differentiation and increases the proportion of multipotent progenitors. Furthermore, we demonstrate for the first time that avian tripotent glial, melanocyte and smooth muscle NC progenitors, as well as bipotent melanocyte and smooth muscle NC progenitors, are capable of self-renewal. FGF2 significantly stimulated the self-renewal of bipotent progenitor cells with glial cells and SMC potentials. These cells propagated for many generations and behaved as stem cells. These results suggest an important role of FGF2 in maintaining the stemness of avian NC cells.