Upon immune cell activation with antigen, growth factors, or other stimuli, the cytoskeleton undergoes extensive reorganization to elicit a cellular response. The cytoskeleton, consisting of microtubules and actin, is a highly organized network regulated by various signal transduction pathways. Specifically, Rho GTPases (RhoA, Rac1 and Cdc42) regulate the cytoskeleton, albeit through different pathways. p21-activated kinases (Pak) are serine/threonine kinases directly bound and activated by Rac1 and Cdc42. There are 6 Pak isoforms separated into 2 groups (groups I&II) in this family of kinases, and only recently have isoform specificities been identified by the use of genetically-engineered mouse models deleted for individual isoforms. In this dissertation we sought to identify if differences exist between Pak1 and Pak2 in immune function, in particular how they differ in regulation of the cytoskeleton reorganization required for immune cell function. Using primary bone marrow derived mast cells, an immune cell type responsible for anaphylaxis and allergic responses, we identified that Pak1 and Pak2 function in opposing manners with regard to antigen-induced degranulation. We identified key mechanisms involved in Pak2's negative regulation of mast cell degranulation. These findings identify potential therapeutic side effects with the use of recently developed pan-Pak inhibitors in the clinic. Pak2 deletion was additionally investigated in an in vivo mouse model. We discovered that Pak2 is critical for homeostasis and survival in an adult animal. We identified macrothrombocytopenia, cause by an increase in circulating platelet half-life and clearance, as well as other defects in Pak2-deleted adult mice. Therefore, we evaluated the maturation process of the platelet-producing megakaryocyte and found that Pak2-null megakaryocytes have altered microtubules, proplatelet extensions and polyploidization. Various signaling pathways that regulate these functions were also suppressed with Pak2 deletion. Together, our findings identify Pak2 as the predominant isoform in hematopoietic compartment and immune cells, and suggest further analysis of critical immune cell side effects, which could occur in the patient with the use of pan-Pak inhibitors in the treatment of various cancers.