Three characteristics distinguish the six layers of the adult tree shrew dorsal lateral geniculate nucleus (LGN). First, interlaminar spaces divide the nucleus into cell layers. Second, input from the two eyes projects to the nucleus such that two layers (1 and 5) receive input from the ipsilateral retinal and four layers (2, 3, 4, and 6) receive input from the contralateral retina. Finally, distinct cytological features characterize individual layers. In this report, we describe the postnatal development of LGN layers in the tree shrew in terms of the development of these three characteristics. At birth, the nucleus appears homogenous in Nissl-stained sections. Thus, no interlaminar spaces are present and all cells look similar in shape, size, and staining intensity. However, autoradiographic data show that, at birth, retinal afferents are segregated in an adult-like pattern. Interlaminar spaces begin to be evident between layers innervated by opposite eyes on postnatal day 2. Several days later, interlaminar spaces between layers innervated by the same eye (i.e., the borders of layer 3) appear, while the others continue to widen. Although some cytological maturation begins before interlaminar space formation, it is not until interlaminar spaces are apparent that features such as differential staining intensity and cell size can be used to distinguish individual layers. The results suggest that the three characteristics that define LGN layers in the tree shrew may be temporally separate events in the developing nucleus. Thus, retinal afferents are segregated prior to interlaminar space formation which, in turn, is initiated prior to final maturation of the cytological features that characterize the cell layers. This may indicate a degree of developmental independence among these maturational events.