The Ebf transcription factors play important roles in the developmental processes of many tissues. We have shown previously that four members of the Ebf family are expressed during mouse retinal development and are both necessary and sufficient to specify multiple retinal cell fates. Here we describe the changes in cell differentiation and retinal ganglion cell (RGC) projection in Ebf1 knockout mice. Analysis of marker expression in Ebf1 null mutant retinas reveals that loss of Ebf1 function causes a significant increase of Müller cells. Moreover, there is an obvious decrease of ipsilateral and retinoretinal projections of RGC axons at the optic chiasm, whereas the contralateral projection significantly increases in the mutant mice. These data together suggests that Ebf1 is required for suppressing the Müller cell fate during retinogenesis and important for the correct topographic projection of RGC axons at the optic chiasm.