The thalamocortical (TC) projection in the mammalian brain is a well characterized system in terms of laminar specificity of neocortical circuits. To understand the mechanisms that underlie lamina-specific TC axon targeting, we studied the role of extracellular and cell surface molecules that are expressed in the upper layers of the developing cortex in in vitro culture techniques. The results demonstrated that multiple upper layer molecules co-operated to produce stop behaviour of TC axons in the target layer. Activity dependency of TC axon branching was also investigated in organotypic co-cultures of the thalamus and cortex. TC axon branches were formed dynamically by addition and elimination during the second week in vitro, when spontaneous firing increased in thalamic and cortical cells. Pharmacological blockade of firing or synaptic activity reduced the remodelling process, in particular branch addition, in the target layer. Together, these findings suggest that TC axon targeting mechanisms involve the regulation with multiple lamina-specific molecules and modification of the molecular mechanisms via neural activity.