Stem cell niches provide a microenvironment to support the self-renewal and multi-lineage differentiation of stem cells. Cell-cell interactions within the niche are essential for maintaining tissue homeostasis. However, the niche cells supporting mesenchymal stem cells (MSCs) are largely unknown. Using single-cell RNA sequencing, we show heterogeneity among Gli1+ MSCs and identify a subpopulation of Runx2+/Gli1+ cells in the adult mouse incisor. These Runx2+/Gli1+ cells are strategically located between MSCs and transit-amplifying cells (TACs). They are not stem cells but help to maintain the MSC niche via IGF signaling to regulate TAC proliferation, differentiation, and incisor growth rate. ATAC-seq and chromatin immunoprecipitation reveal that Runx2 directly binds to Igfbp3 in niche cells. This Runx2-mediated IGF signaling is crucial for regulating the MSC niche and maintaining tissue homeostasis to support continuous growth of the adult mouse incisor, providing a model for analysis of the molecular regulation of the MSC niche. Keywords: IGF signaling; Runx2; adult mouse incisor; growth rate; mesenchymal stem cells; niche; transit-amplifying cells.