The establishment of topographic retinotectal connections requires precise regulation of axonal growth. EphA3 stimulates axon growth of nasal retinal ganglion cells (RGCs) towards the caudal tectum, inhibiting branching in the rostral tectum. GDNF and BDNF stimulates RGC axon growth, though its effects on RGC growth cone dynamics are unclear. We examined the individual and combined effects of EphA3, GDNF and BDNF on growth cone dynamics of dissociated nasal retinal ganglion cells (RGCs) from chicken embryos. Dissociated RGCs were cultured and exposed to EphA3-Fc, GDNF, BDNF, or EphA3-Fc combined with neurotrophic factors. Growth cone velocity, orientation and persistence were quantified by timelapse imaging in Dunn’s chamber. EphA3 gradients significantly increased axon extension rate and promoted directional attraction. EphA3 gradients and neurotrophic factors (notably GDNF) promoted axon extension and orientation; combined EphA3+GDNF synergistically accelerated growth, stabilized trajectories, and reduced exploratory behavior. These results indicate that growth dynamics are critically modulated by guidance cues and trophic support, and that their combined action promotes robust, directed outgrowth. Our findings provide novel insight into mechanisms shaping retinotectal map formation and suggest that synergistic interactions between guidance molecules and neurotrophic factors could inform strategies to enhance axonal regeneration. Support: UBACYT0197BA.