AbstractAscending somatosensory pathways are crossed pathways representing each side of the body in the contralateral neocortex. The principal sensory nucleus of the trigeminal nerve (PrV) relays the facial sensations to the contralateral somatosensory cortex via the ventrobasal thalamus. In the companion article (Kivrak and Erzurumlu [2012] J. Comp. Neurol. 12–0013) we described the normal development of the trigeminal lemniscal pathway in the mouse. In this study we investigated the role of midline axon navigation signals, the netrin and slit proteins. In situ hybridization assays revealed that both netrin and slit mRNAs are expressed along the midline facing the PrV axons and their receptors are expressed in developing PrV neurons. In wild‐type mouse embryos, PrV axons cross the midline and take a sharp rostral turn heading toward the contralateral thalamus. Examination of trigeminal lemniscal axons in dcc knockout mice revealed absence of midline crossing between E11 and E15. However, a few axons crossed the midline at E17 and reached the contralateral thalamus, resulting in a bilateral PrV lemniscal pathway at P0. We also found that slit1, ‐2 or ‐3 single or double knockout mice have impaired development of the trigeminal–lemniscal pathway. These include axon stalling along the midline, running within the midline, and recrossing of axons back to the site of origin. Collectively, our studies indicate a cooperative role for netrin and slit proteins in midline attraction and crossing behavior of the ascending facial somatosensory projections during development. J. Comp. Neurol. 521:312–325, 2013. © 2012 Wiley Periodicals, Inc.