Abstract Hematopoietic stem (HSC) and progenitor (HPC) cells are regulated by interacting signals and cellular and noncellular elements of the hematopoietic niche. We previously showed that CD166 is a functional marker of murine and human HSC and of cellular components of the murine niche. Selection of murine CD166+ engrafting HSC enriched for marrow repopulating cells. Here, we demonstrate that CD166-CD166 homophilic interactions enhance generation of murine and human HPC in vitro and augment hematopoietic function of these cells. Interactions between cultured CD166+ Lineage−Sca-1+c-Kit+ (LSK) cells and CD166+ osteoblasts (OBs) significantly enhanced the expansion of colony-forming units (CFUs). Interactions between CD166+ LSK cells and immobilized CD166 protein generated more CFU in short-term cultures than between these cells and bovine serum albumin (BSA) or in cultures initiated with CD166− LSK cells. Similar results were obtained when LSK cells from wildtype (WT) or CD166 knockout (KO) (CD166−/−) mice were used with immobilized CD166. Human cord blood CD34+ cells expressing CD166 produced significantly higher numbers of CFUs following interaction with immobilized CD166 than their CD166− counterparts. These data demonstrate the positive effects of CD166 homophilic interactions involving CD166 on the surface of murine and human HPCs. Single-cell RNA-seq analysis of CD150+CD48− (signaling lymphocyte activation molecule (SLAM)) LSK cells from WT and CD166−/− mice incubated with immobilized CD166 protein revealed that engagement of CD166 on these cells activates cytokine, growth factor and hormone signaling, epigenetic pathways, and other genes implicated in maintenance of stem cell pluripotency-related and mitochondria-related signaling pathways. These studies provide tangible evidence implicating CD166 engagement in the maintenance of stem/progenitor cell function. Stem Cells 2019;37:1319–1330 Significance Statement This study describes how homophilic interactions via CD166 that is expressed on hematopoietic cells augments the functional potential of these cells. This study also uses single-cell RNAseq to identify pathways activated by the engagement of CD166 on the surface of these cells to enhance the hematopoietic function of stem and progenitor cells.