Early life microbial exposure can permanently program the offspring’s immune system and life-long disease risk. For example, children exposed to farm environments are less likely to develop asthma, and antibiotic use in early life is associated with an increased risk of developing inflammatory bowel disease and diabetes. Recent studies have also observed a correlation between cesarean section birth and diabetes, asthma and allergic disorders later in life. However, these studies are based on epidemiological associations, and the underlying mechanisms remain undefined. In this administrative supplement, we will leverage a unique pet-shop ‘dirty’ mouse model to determine how the microbial environment alters the generation of immune cells from hematopoietic stem cells (HSCs). Our preliminary data indicates that adult mice raised in a dirty environment are more resistant to infection because they contain more ‘fast-acting’ lymphocytes made by fetal HSCs and fewer ‘slower-acting’ lymphocytes made by adult HSCs. However, all of our studies to date have been performed with mature lymphocytes, and we have yet to explore the impact of the microbial environment on the HSC compartment. Thus, we are seeking funds to use a single-cell RNA-seq platform to examine how the microbial environment alters the transition from fetal to adult hematopoiesis. Knowledge gained from these studies will provide key insights into how the early microbial environment leads to permanent changes in immune development and later-life susceptibility to disease.