It has been shown in vivo that Wnt5a gradients surround the corpus callosum and guide callosal axons after the midline (postcrossing) by Wnt5a-induced repulsion via Ryk receptors. In dissociated cortical cultures we showed that Wnt5a simultaneously promotes axon outgrowth and repulsion by calcium signaling. Here to test the role of Wnt5a/calcium signaling in a complex in vivo environment we used sensorimotor cortical slices containing the developing corpus callosum. Plasmids encoding the cytoplasmic marker DsRed and the genetically encoded calcium indicator GCaMP2 were electroporated into one cortical hemisphere. Postcrossing callosal axons grew 50% faster than pre-crossing axons and higher frequencies of calcium transients in axons and growth cones correlated well with outgrowth. Application of pharmacological inhibitors to the slices showed that signaling pathways involving calcium release through IP3 receptors and calcium entry through TRP channels regulate post-crossing axon outgrowth and guidance. Co-electroporation of Ryk siRNA and DsRed revealed that knock down of the Ryk receptor reduced outgrowth rates of postcrossing but not precrossing axons by 50% and caused axon misrouting. Guidance errors in axons with Ryk knockdown resulted from reduced calcium activity. In the corpus callosum CaMKII inhibition reduced the outgrowth rate of postcrossing (but not precrossing) axons and caused severe guidance errors which resulted from reduced CaMKII-dependent repulsion downstream of Wnt/calcium. We show for the first time that Wnt/Ryk calcium signaling mechanisms regulating axon outgrowth and repulsion in cortical cultures are also essential for the proper growth and guidance of postcrossing callosal axons which involve axon repulsion through CaMKII.