Abstract: Background　Current systematic understanding of the hematopoietic microenvironment in the human fetal liver remains inadequate. In particular, there is a lack of in-depth investigation into the regulatory differences in hematopoietic stem cell (HSC) engraftment before and after colonization of the fetal liver.Objective　To investigate the heterogeneity of human fetal liver HSCs at the single-cell transcriptome level, characterize the fetal liver hematopoietic microenvironment before and after HSC homing, and identify key molecular mechanisms potentially regulating distinct HSC functions.Methods　Single-cell transcriptomic data from human fetal livers (CS14–CS23) were integrated and analyzed. Data integration, UMAP dimensionality reduction, and unsupervised clustering (resolution 0.2) were performed using Seurat. Differentially expressed genes were identified using FindAllMarkers (Wilcoxon test, log2FC＞1.5, P.adjust＜0.05). GO enrichment analysis was conducted with clusterProfiler, and cellcell communication was inferred using CellChat. Results　(1) A comprehensive map of the fetal liver hematopoietic microenvironment before and after HSC homing was generated. Cell-cell interaction analysis between HSCs and niche cells revealed that CD99, which is highly expressed in mesenchymal and endothelial cells, may promote HSC homing. (2) Human fetal liver HSCs exhibited heterogeneity, comprising an HSC1 subset with stronger stemness and relatively higher CD99 expression, and an HSC2 subset with active cell cycle. (3) Both HSC1 and HSC2 subsets receive stemness-maintaining intercellular signals from the hepatic niche, including pleiotrophin (PTN), osteopontin (SPP1), and endothelial cell adhesion molecule (ESAM). Specifically, the HSC1 subset is uniquely enriched for protein C (PROC) signaling to sustain stem cell identity, whereas the HSC2 subset exhibits specific enrichment of protease-activated receptor (PAR) signaling to drive cellular proliferation.Conclusion　This study reveals that CD99 in the human fetal liver hematopoietic microenvironment may facilitate HSC homing and elucidates the heterogeneous mechanisms by which the stemness-maintaining HSC1 subset and the proliferative HSC2 subset regulate stemness and proliferation through PROC and PAR signaling, respectively.