Neurotrophic factors present as concentration gradients are neurotropic cues that direct axonal growth toward their targets. Multiple factors work together in vivo to ensure axons reach the proper targets, likely interacting with one another via intracellular signalling pathways. Nerve growth factor (NGF) and neurotrophin-3 (NT-3) are neurotrophins known to guide axons as well as promote axonal growth following injury to both the spinal cord and peripheral nerve. These molecules interact with neurons through different tyrosine kinase receptors. In this study, the receptors for these growth factors were shown to be co-localized on E10 chick dorsal root ganglion (DRG) cells, providing an opportunity for synergism. Well-defined concentration gradients of NGF and NT-3 were immobilized for the first time in a cell-penetrable, cell-adhesive scaffold of poly(2-hydroxyethylmethacrylate) and poly(L-lysine). An NGF concentration gradient of 310 ng/mL/mm was required to guide chick DRG neurites. A lower concentration gradient of 200 ng/mL/mm of NGF was shown to elicit guidance when an NT-3 concentration gradient of 200 ng/mL/mm was also present, indicating a synergistic response in the DRG neurons. These gradient scaffolds may be useful for guided regeneration following injury to the spinal cord or peripheral nerve and may also elucidate the mechanism for intracellular signaling of neurotrophic factors.