The extracellular signal-regulated kinase 5 (ERK5) is activated in neurons of the central nervous system by neurotrophins including brain-derived neurotrophic factor (BDNF). Although MEK5 is known to mediate BDNF stimulation of ERK5 in central nervous system neurons, other upstream signaling components have not been identified. Here, we report that BDNF induces a sustained activation of ERK5 in rat cortical neurons and activates Rap1, a small GTPase, as well as MEKK2, a MEK5 kinase. Our data indicate that activation of Rap1 or MEKK2 is sufficient to stimulate ERK5, whereas inhibition of either Rap1 or MEKK2 attenuates BDNF activation of ERK5. Furthermore, BDNF stimulation of MEKK2 is regulated by Rap1. Our evidence also indicates that Ras and MEKK3, a MEK5 kinase in non-neuronal cells, do not play a significant role in BDNF activation of ERK5. This study identifies Rap1 and MEKK2 as critical upstream signaling molecules mediating BDNF stimulation of ERK5 in central nervous system neurons.