RT4 is a family of cell lines derived from a rat peripheral neurotumor and consists of a multipotential stem cell which spontaneously gives rise to a glial derivative and two neuronal derivatives. To begin to understand the role(s) of transcription factors in neural differentiation, we examined the expression of ten transcription factor genes (MASH1, REST/NRSF, Oct-1, Oct-2, Tst-1/SCIP, Brn-1, Brn-2, Brn-3.0, Brn-4, Brn-5) in the RT4 cell lines. We report here that all of the RT4 cells express REST/NRSF, Oct-1 and Brn-5, but do not express MASH1, Brn-3.0 or Brn-4. Furthermore, Brn-2 and Tst-1/SCIP expression was restricted to the RT4 stem cell line and glial derivative, while Oct-2 was expressed predominantly by the RT4 stem cell line and neuronal derivatives. We propose that the lack of expression of MASH1 (which is expressed relatively early in autonomic neuron differentiation) and Brn-3.0 (which is expressed early in sensory neuron differentiation), in combination with the presence of REST/NRSF (a repressor of neuronal gene expression), in all of the RT4 cell lines, establishes the RT4 system as a unique model for examining very early events in neuronal versus glial cell fate determination.