EphB receptor tyrosine kinases regulate cell-cell contacts throughout nervous system development, mediating processes as diverse as axon guidance, topographic mapping, neuronal migration and synapse formation. EphBs bind to a group of ligands, ephrin-Bs, which span the plasma membrane, thus allowing for bidirectional signaling between cells. Since EphBs are capable of multiple modes of signaling, and since they regulate numerous interdependent stages of development, it has been challenging to define which signaling functions of EphBs mediate particular developmental events. To overcome this hurdle, we developed an approach combining chemical biology with genetic engineering to reversibly inhibit EphB receptors in vivo. By mutating a residue in the receptor’s ATP-binding pocket, we rendered its kinase activity sensitive to reversible inhibition by PP1 analogs that do not inhibit wild type receptors. We engineered triple knockin mice bearing this mutation in which the kinase activity of EphB1, EphB2, and EphB3 can be rapidly, reversibly, and specifically blocked. Since we are able to block the kinase activity of EphBs while leaving their scaffolding and reverse signaling capabilities intact, we can precisely isolate the role of the kinase domain. In addition, acute inhibition can circumvent the developmental compensation that may occur after genetic mutations and can even allow the controlled study of EphBs in the mature brain and in disease models. Using these mice, termed analog-sensitive EphB triple knockin (AS-EphB TKI) mice, we demonstrate a requirement for the kinase-dependent signaling of EphBs in the collapse of retinal ganglion cell growth cones in vitro and the guidance of retinal axons at the optic chiasm in vivo. In addition, we show that the formation of several cortical axon tracts, including the corpus callosum, requires EphB tyrosine kinase signaling. In contrast, we find that steps in synapse development that are thought to be EphB-dependent occur normally when the kinase activity of EphBs is inhibited. We conclude that a cardinal in vivo function of EphB signaling, the ability to mediate axon guidance via growth cone repulsion, requires the tyrosine kinase activity of EphBs, while the development of functional excitatory synapses is independent of EphB tyrosine kinase activity.