The immune system employs a “double-edged sword” approach to combat infections: the innate and the adaptive immune responses. The innate immune response provides a rapid, “non-specific” first line of defense, while the adaptive immune response tailors a more targeted response to specific pathogens. B-2 cells are part of the adaptive immune response, generating high-affinity antibodies after encountering a foreign antigen. In contrast, B-1 cells, B lymphocytes with innate-like B qualities, bridge the gap between innate and adaptive immunity. They differ from B-2 cells in development, phenotype, localization, and function. B-1 cells are generated in ontogeny during the late fetal and early neonatal period. B-1 cell-derived plasma cells in spleen and bone marrow are the main producers of natural IgM, which are produced even in the absence of foreign antigen exposure. Identifying genes critical for B-1 cell development and/or maintenance would improve our understanding of how natural IgM production is regulated. In Chapter 1, I explore key molecules and genes that have been identified to be associated with B-1 cell development, maturation, differentiation, regulation, and migration. I aim to illuminate the intricate molecular landscape governing B-1 cells, pinpointing areas where further exploration is necessary to advance our understanding of these crucial players of the immune system. In Chapter 2, I establish and implement a strategy to analyze mouse spleen samples from 652 distinct mouse strains, each carrying a single gene knock out mutation (KO), by flow cytometry, using two multicolor panels with which 57 distinct leukocyte subsets are identified, and analyze to collect the data into a searchable database. In Chapter 3, I identify candidate genes critical for B-1 cells development, maintenance, and/or differentiation utilizing the Mouse Spleen Immunophenotyping Database. I generate and curate data from all tested mouse strains and identified seven genes that specifically affected B-1 cells in both males and females, none had been associated previously with B-1 cells. Among them was Ppp2r5a, a gene I identified for further analysis. Flow cytometric analysis of Ppp2r5a deficient mice revealed sex-specific B-1 cell differences. The work outlined in this dissertation describes how we utilized an unbiased approach at identifying novel genes that affect B-1 cell development and/or maintenance. Together, the studies identify Ppp2r5a, a subunit of the protein phosphatase 2A, as a novel gene selectively regulating the development and/or maintenance of B-1 cells.