To develop a functional nervous system, neural circuits are initially established through the stepwise process of neural specification, axon guidance and synaptogenesis. Axon guidance, the process by which neurons extend axons to their final targets, relies on the presence of extracellular cues, and their respective guidance receptors. Intracellular molecules that transduce these signals are currently unidentified for many guidance receptors. We are particularly interested in identifying the signal transduction mechanisms that underlie attractive growth cone turning in response to the the conserved axon guidance cue, Netrin. Netrin elicits midline axon attraction in all bilaterally symmetric animals studied to date through the DCC family of receptors. In Drosophila, commissural neurons require Netrin and its DCC receptor, Frazzled, for midline axon crossing. To identify genes that function in Netrin signal transduction in vivo, we have taken two approaches: (1) a candidate gene approach to address a well-accepted model based on in vitro observation and (2) a genetic screen to identify novel genes that regulate commissural axon guidance. We find here that two different tyrosine kinases, Src and Abl, regulate commissural guidance, but through different mechanisms. Src, in contrast to the prevailing view, is not required for Netrin signaling in commissural neurons and instead antagonizes midline axon crossing through a novel pathway. Abl promotes Netrin-dependent attraction through its C-terminal scaffolding domain through a mechanism that is partly kinase-independent. Additionally, we have identified several new genomic loci that contribute to commissural guidance in vivo.