EphB tyrosine kinase receptors have been implicated in multiple developmental processes; however, the signaling mechanism underlying these events remains unclear. Through a triple knock-in mouse line for three neurally expressed EphBs, Sokis et al. demonstrated that EphB tyrosine kinase activity is required for axon guidance but does not influence synapse formation. This short communication highlights their study and appealing molecular approach that elucidated the functions of EphB tyrosine kinase during developmental events.