Smoking is the leading cause of preventable illness and death in the United States. Despite this, current pharmacological treatments for nicotine withdrawal remain partially effective and the abstinence rate remains low. The development of novel pharmacotherapies that can mediate the aversive symptoms of nicotine withdrawal will contribute to improved cessation outcomes. To this end, the studies in this dissertation had two objectives: one- to identify novel molecular pathways underlying nicotine withdrawal and two- to examine the contribution of genetic variants to withdrawal symptoms. To address the first objective we used functional genomic approaches (chromatin immunoprecipitation and whole genome sequencing (ChIP-Seq)) to identify pathways that are altered following withdrawal from nicotine. Through these means we identified the AMPK (AMP-activated protein kinase) signaling pathway as one such target. In addition, we demonstrated that the AMPK pathway is activated following chronic nicotine, an effect that is reversed by 24-hour withdrawal. Maintaining AMPK activation during withdrawal via the preclinical AMPK activator AICAR or the FDA approved diabetes drug metformin attenuated anxiety-like behavior during nicotine withdrawal in preclinical animal models. Further, the reduction in anxiety-like behavior was through a mechanism dependent on the AMPKα subunit within the hippocampus, as determined by a genetic AMPKα loss-of function animal model. Metformin treatment also reversed withdrawal-induced deficits in contextual fear conditioning. In our final study we examined the contribution of genetic variants to the affective phenotypes of nicotine withdrawal. We utilized a BDNF knock-in mouse containing the BDNF Val66Met polymorphism to examine the effect of this single nucleotide polymorphism (SNP) on anxiety-like behavior following withdrawal. Using this model we found blunted anxiety-like behavior in mice homozygous for the SNP (BDNFMet/Met) following withdrawal. We also observed an increase in expression of the BDNF prodomain in BDNFMet/Met mice following nicotine withdrawal, highlighting the potential role of this ligand in mediating withdrawal behavior. Together, research from this dissertation provide the first evidence of a direct effect of AMPK modulation on behavior following nicotine withdrawal, and provides the first example of using a mouse genetic variant model to examine withdrawal behavior.