Neurons generated early in development of the ventral diencephalon have been shown to play a key role in defining the midline and the caudal boundary of the optic chiasm in the mouse retinofugal pathway. These functions have been attributed to a surface bound adhesion molecule, CD44 that is expressed in these chiasmatic neurons. In this study, we investigated the effects of perturbing normal CD44 functions on axon routing in brain slice preparations of the mouse retinofugal pathway. Two CD44 antibodies (Hermes-1 and IM7) were used that bind to distinct epitopes on the extracellular domain of the molecule. We found that both antibodies produced dramatic defects in routing of the retinal axons that arrive early in the chiasm. In preparations of embryonic day 13 (E13) and E14 pathways, the crossed component in the chiasm was significantly reduced after antibody treatment. However, such reduction in axon crossing was not observed in E15 chiasm, indicating that the lately generated crossed axons lost their responses to CD44. Furthermore, the anti-CD44 treatment produced a reduction in the uncrossed component in the E15 but not in younger pathways, suggesting a selective response of the lately generated axons, mostly from ventral temporal retina, but not those generated earlier, to the CD44 at the chiasmatic midline in order to make their turn for the uncrossed pathway. These findings provide evidence that a normal function of CD44 molecules in the chiasmatic neurons is essential for axon crossing and axon divergence at the mouse optic chiasm.