In addition to regulating mast cell homeostasis, the activation of KIT following ligation by stem cell factor promotes a diversity of mast cell responses, including cytokine production and chemotaxis. Although we have previously defined a role for the mammalian target of rapamycin complex 1 in these responses, it is clear that other signals are also required for maximal KIT-dependent cytokine production and chemotaxis. In this study, we provide evidence to support a role for glycogen synthase kinase 3β (GSK3β) in such regulation in human mast cells (HuMCs). GSK3β was observed to be constitutively activated in HuMCs. This activity was inhibited by knockdown of GSK3β protein following transduction of these cells with GSK3β-targeted shRNA. This resulted in a marked attenuation in the ability of KIT to promote chemotaxis and, in synergy with FcεRI-mediated signaling, cytokine production. GSK3β regulated KIT-dependent mast cell responses independently of mammalian target of rapamycin. However, evidence from the knockdown studies suggested that GSK3β was required for activation of the MAPKs, p38, and JNK and downstream phosphorylation of the transcription factors, Jun and activating transcription factor 2, in addition to activation of the transcription factor NF-κB. These studies provide evidence for a novel prerequisite priming mechanism for KIT-dependent responses regulated by GSK3β in HuMCs.