The Brn-3 subfamily of POU domain transcription factors consists of Brn-3a, -3b, and -3c, which are important regulators for sensorineural development. Despite the expression of all three factors in retinal ganglion cells, earlier studies have shown that Brn-3b is the only one among the three Brn-3 genes that is essential for development of approximately 70% of ganglion cells in the murine retina. I report here that Brn-3b displays a spatiotemporal expression pattern characteristic of the dynamic profile of ganglion cell genesis during murine retinal development. Moreover, it is initially turned on in postmitotic ganglion cell precursors 2 days before the onset of Brn-3a and -3c expression in differentiated ganglion cells. During the entire period of retinal ganglion cell genesis, the postmitotic ganglion cell precursors that would normally become Brn-3b+ cells fail to properly differentiate in Brn-3b-/- mice, as evidenced by a twofold reduction in the optic nerve size and diminished expression of several ganglion cell markers. The undifferentiated ganglion cell precursors appear to be degenerated by apoptosis within the ganglion cell layer during the perinatal and early postnatal period. I propose that retinal ganglion cells develop following two separate differentiation pathways--Brn-3b dependent and Brn-3b independent. In the Brn-3b-dependent mechanism, Brn-3b may be required to initiate a particular differentiation program for a large set of postmitotic ganglion precursors to properly differentiate into the 70%, Brn-3b-dependent retinal ganglion cells.