We demonstrated in a previous study that, following neonatal bilateral enucleation in the tree shrew, interlaminar spaces (ILSs) in the dorsal lateral geniculate nucleus fail to form. In the present study we sought to determine which aspects of ILS formation are dependent upon retinal input. Accordingly, we studied the degree of ILS formation in tree shrews which were bilaterally enucleated either during ILS formation on postnatal day 3 (P3) or just after all ILS were apparent but before they had reached a mature width (P15). Our results indicate that retinal input is necessary for the initial formation of each ILS, but that it is not required for the maturation or maintenance of ILSs which have already begun to form.