Immune checkpoint blockade has revolutionized cancer treatment but has only benefited a subset of patients. Among the variables that could contribute to interpatient heterogeneity is differential composition of the patients’ microbiome, which has been shown to affect anti-tumor immunity and immunotherapy efficacy in preclinical mouse models. We began by investigating whether there is an association between response to anti-PD-1 therapy and the composition of the gut microbiome (Chapter 3). We analyzed baseline stool samples from metastatic melanoma patients before immunotherapy treatment through an integration of 16S ribosomal RNA gene sequencing, metagenomic shotgun sequencing, and quantitative polymerase chain reaction for selected bacteria. A significant association was observed between commensal microbial composition and clinical response. Reconstitution of germ-free mice with fecal material from responding patients could lead to improved tumor control, augmented T cell responses, and greater efficacy of anti-PD-L1 therapy. These results suggest that the commensal microbiome may have a mechanistic impact on anti-tumor immunity in human cancer patients. Next, we explored the cellular mechanisms responsible for variable responses to cancer immunotherapy using mice stably colonized by patient commensals (Chapter 4). We rigorously evaluated the tumor immune infiltrate in representative responder and non-responder models using single cell gene expression, flow cytometry and functional tests to identify the source of resistance to immune checkpoint blockade. We found that variable tumor control with anti-PD-L1 was CD8+ T cell-dependent, and likely stemmed from distinct activation states of myeloid cells within the tumor microenvironment. Collectively, these results illustrate how the gut microbiome can impact the tumor microenvironment by tuning innate immunity and offer direction for future studies investigating the link between commensal bacteria and immunotherapy efficacy in patients.