Hematopoietic stem cell transplantation (HSCT) cures a range of fatal conditions including immunodeficiency, bone marrow (BM) failure, and malignancy, and more than 20,000 HSCTs occur annually in the U.S. Decades of research have reduced post-transplant morbidity and improved survival, but barriers remain substantial including the need for optimal engraftment of donor stem cells. Indeed, knowledge about the genetic and molecular constituents necessary for HSC homeostasis and post-transplant engraftment remain incomplete. We have identified that follistatin-like 1 (FSTL-1) is critical for multilineage reconstitution following HSCT, but the mechanism remains unknown. Transcriptionally, FSTL-1 is most highly expressed in the rare population of self-renewing Long-term HSC (LT-HSC). Our preliminary data and reports from the literature lead us to hypothesize that Fstl1 is critical for both HSC homeostasis and post-transplant cellular engraftment through its promotion of LT-HSC quiescence. This idea will be tested in two Specific Aims. Aim 1 will determine the impact of FSTL-1 deficiency on HSC homeostasis. Using a FSTL-1 conditional knock-out (CKO) model, we will determine which HSC populations and cellular pathways are impacted by FSTL-1 deficiency under homeostatic conditions as well as the response of FSTL-1 deficient cells to 5-flurouracil induced cell-cycling toxicity. Aim 2 will determine the impact of FSTL-1 modulation on HSC and bone marrow engraftment. Here, using FSTL-1 CKO cells, we will determine the impact of FSTL-1 deficiency directly in a highly purified LT-HSCs population and will determine FSTL-1 role in HSC self-renewal using serial transplantation and in vitro colony-forming assays. Separately, we will develop a lentiviral system for ectopic FSTL-1 expression, followed by efforts to rescue the diminished engraftment of FSTL-1-deficient cells. Finally, we will test whether lentiviral-driven FSTL-1 expression can enhance HSCT engraftment independent of FSTL-1 deficiency. Cumulatively, the proposed studies will focus on defining the role of FSTL-1 in HSC homeostasis and immune reconstitution. The findings will benefit the fields of stem cell biology, immune system development, and preclinical modeling of cellular transplantation, as well as the study of FSTL-1 cellular and molecular signaling. Ultimately, this work will inform novel approaches to improve human disease in the fields of primary immunodeficiency and impaired HSC function, and blood and marrow transplantation.