During development, mitral cell axons, the major efferents of the olfactory bulb, exhibit a protracted waiting period in the lateral olfactory tract (LOT) before giving off collateral branches and innervating the target olfactory cortex. To investigate the target invasion mechanism, a series of heterochronic and heterotopic cocultures of olfactory bulbs with various olfactory cortical strips were conducted. These experiments indicated that development of collateral branches is triggered by environmental cues but not by intrinsic mechanisms in mitral cells. The collateral-inducing cues are apparently different from the cues directing outgrowth of primary mitral cell axons. Coculture experiments also indicated that the target olfactory cortex undergoes a developmental change to become accessible to mitral cell fibers. Primary mitral cell axons, however, still preferred the LOT position over such accessible piriform cortex when encountered both the locations. These results suggest that mitral cell projection comprises multiple steps which are controlled by various environmental cues.