Nanoparticles (NPs) are promising vehicles for drug delivery because of their potential to target specific tissues . Although it is known that NP size plays a critical role in determining their biological activity, there are few quantitative studies of the role of NP size in determining biodistribution after systemic administration. Here, we engineered fluorescent, biodegradable poly(lactic-co-glycolic acid) (PLGA) NPs in a range of sizes (120–440 nm) utilizing a microfluidic platform and used these NPs to determine the effect of diameter on bulk tissue and cellular distribution after systemic administration. We demonstrate that small NPs (∼120 nm) exhibit enhanced uptake in bulk lung and bone marrow, while larger NPs are sequestered in the liver and spleen. We also show that small NPs (∼120 nm) access specific alveolar cell populations and hematopoietic stem and progenitor cells more readily than larger NPs. Our results suggest that size of PLGA NPs can be used to tune delivery to certain tissues and cell populations in vivo.