Differentiation from hematopoietic stem and progenitor cells (HSPCs) to committed blood lineages is orchestrated by a network of transcription factors and cytokines that promote lineage specific gene expression while simultaneously repressing genes associated with alternative lineages. In addition to transcription factors and cytokines, small non-coding microRNAs (miRNAs) also have the potential to influence cell fate decisions through negative regulation of lineage specific genes. The aim of this dissertation was to elucidate the role of two microRNA clusters, mirn23a and mirn23b, as well as the transcription factor Arid3b, during hematopoietic development in mice. In chapter 1, I characterize a mirn23a germline knockout mouse and observe that loss of mirn23a results in increased B lymphopoiesis at the expense of myelopoiesis, which is the first report of a miRNA being necessary for immune cell fate decisions in a genetic knockout model. In chapter 2, I elucidate the molecular mechanisms by which mirn23a influences cell fate decisions and report that mirn23a negatively regulates essential B cell transcriptional networks to promote myeloid development. Chapter 3 focuses on the effect of compound loss of both mirn23a and mirn23b miRNA clusters using a conditional knockout system, which revealed that loss of both mirn23a and mirn23b results in decreased HSPC production and bone marrow cellularity. For the final chapter, I characterize mice deficient for transcription factor Arid3b using a conditional knockout system and observe that Arid3b is necessary for de novo B cell production. Together, this work enhances our understanding of the molecular mechanisms governing hematopoiesis.