The stem cell niche is the microenvironment where stem cells reside and receive specific cues. These cues can be cell to cell or cell to extracellular matrix (ECM) interactions and signals in which they influence the stem cell behavior, determine its fate, and facilitate asymmetric divisions [1]. This process remains poorly understood, highlighting the need for improved model systems. To address this, we present a novel 3D bioprinting system that enables the study of this niche at a single-cell level [2]. We demonstrate that the system allows for the printing of single cells in grids and large arrays in both 3D and 2D by injecting into hydrogels or directly onto glass slides using nanoliter droplets. Subsequently, we assessed whether the system could be employed to unravel differential signaling between stem cells and various ECMs. Single mouse embryonic stem cells (mESCs) were printed in different ECMs to uncover the influence of different matrices on the redirection of the stem cell fate. Our results showed that the ESCs fate and proliferation were different when different ECMs were used. We then evaluated if our system could be used to generate artificial niches using immobilized growth factors to drive asymmetric divisions. To achieve this, we conjugated Epidermal growth factor (EGF) and Fibroblast growth factor 2 (FGF-2), growth factors required for the self-renewal of neural stem cells (NSCs), to fluorescent FluoSpheres™. The EGF/FGF tagged FluoSpheres were incubated with NSCs and either cultured or bioprinted into arrays under differentiation conditions. We show that the interaction with the EGF/FGF beads protects NSCs from differentiating using the NSC marker Nestin. Our results therefore demonstrate that the FGF/EGF beads could serve as an artificial niche to protect NSCs from differentiation and drive asymmetric divisions. Collectively, our results demonstrate that our bioprinting system is a unique and robust tool that aids in mimicking the microenvironment where stem cells thrive, while also enabling the study of stem cell-niche interactions at a single-cell level using precision 3D bioprinting techniques. References: 1. Ferraro, F., C.L. Celso, and D. Scadden, Adult stem cels and their niches. Adv Exp Med Biol, 2010. 695: p. 155-68. 2. Reid, J.A., et al., A 3D bioprinter platform for mechanistic analysis of tumoroids and chimeric mammary organoids. Sci Rep, 2019. 9(1): p. 7466.