Fibroblast growth factor 2 (FGF-2) is an important neurotrophic factor that promotes survival of adult mesencephalic dopaminergic (mDA) neurons and regulates their adequate development. Since mDA neurons degenerate in Parkinson's disease, a comprehensive understanding of their development and maintenance might contribute to the development of causative therapeutic approaches. The current analysis addressed the role of FGF-2 in mDA axonal outgrowth, pathway formation, and innervation of respective forebrain targets using organotypic explant cocultures of ventral midbrain (VM) and forebrain (FB). An enhanced green fluorescent protein (EGFP) transgenic mouse strain was used for the VM explants, which allowed combining and distinguishing of individual VM and FB tissue from wildtype and FGF-2-deficient embryonic day (E)14.5 embryos, respectively. These cocultures provided a suitable model to study the role of target-derived FB and intrinsic VM-derived FGF-2. In fact, we show that loss of FGF-2 in both FB and VM results in significantly increased mDA fiber outgrowth compared to wildtype cocultures, proving a regulatory role of FGF-2 during nigrostriatal wiring. Further, we found in heterogeneous cocultures deficient for FGF-2 in FB and VM, respectively, similar phenotypes with wider fiber tracts compared to wildtype cocultures and shorter fiber outgrowth distance than cocultures completely deficient for FGF-2. Additionally, the loss of target-derived FGF-2 in FB explants resulted in decreased caudorostral glial migration. Together these findings imply an intricate interplay of target-derived and VM-derived FGF signaling, which assures an adequate nigrostriatal pathway formation and target innervation. J. Comp. Neurol. 520:3949–3961, 2012. © 2012 Wiley Periodicals, Inc.