A number of signaling molecules and transcription factors play important roles in the development of the autonomic nervous system. Here, we show that mouse trunk neural crest cells can differentiate into autonomic neurons expressing mammalian achaete-scute homolog 1 (mash1), Phox2b, tyrosine hydroxylase, and/or dopamine-beta-hydroxylase in the absence of bone morphogenetic protein (BMP)-4. The expression of mash1 and Phox2b is induced even in the presence of noggin or chordin, which are inhibitors of BMP signaling. Whereas these autonomic neurons do not express c-ret, the receptor for glial-cell-line-derived neurotrophic factor (GDNF), GDNF promotes the differentiation of c-ret-positive autonomic neurons in the presence of noggin. Autonomic neurogenesis is completely prevented by fibroblast growth factor (FGF)-2 treatment or by activation of Notch signaling. Furthermore, the suppression of Phox2b expression by FGF-2 can be recovered by treatment with Notch-1 small interfering RNA. Our data suggest that BMP-independent mechanisms promote the differentiation of autonomic neurons, and that FGF-2 suppresses autonomic neurogenesis by means of the activation of Notch signaling.