We recently described the boundary-like expression pattern of the extracellular matrix molecule tenascin-C (Tnc) in the developing mouse olfactory bulb (OB) (Shay et al., 2008). In the present study, we test the hypothesis that Tnc inhibits olfactory sensory neuron (OSN) axon growth in the developing OB before glomerulogenesis. The period of time before glomerular formation begins, when axons remain restricted to the developing olfactory nerve layer (ONL), is crucial for axon sorting. Here, we show with in vitro analyses that OSN neurite outgrowth is inhibited by Tnc in a dose-dependent manner and that, in stripe assays, axons preferentially avoid Tnc. Using Tnc-null mice, we also show that that glomerular development is delayed in the absence of Tnc. In wild-type mice, OSN axons coalesce into immature or protoglomeruli, which further differentiate and segregate into glomeruli. Glomeruli are first identifiable as discrete structures at birth. In null mice, glomeruli appear immature at birth, remain fused to the ONL, and have a significantly larger diameter compared with wild-type controls. By postnatal day 4, null glomeruli are indistinguishable from controls. Thus, OSN axons appear delayed in their coalescence into glomerular structures. These data correlate with behavioral reports of Tnc-null mice, which are delayed by 24 h in their acquisition of an olfactory behavior (de Chevigny et al., 2006). Collectively, these data demonstrate that Tnc is an inhibitory boundary molecule in the developing OB during a key period of development.