Hematopoietic Stem Cells (HSCs) reside in the bone marrow and generate blood cells throughout life. Our lab recently discovered that HSCs from adult bone marrow (ABM HSCs) are endowed with low intracellular calcium and maintained in low calcium environments both in vitro and in vivo (Luchsinger et al., Cell Stem Cell, 2019). ABM HSCs derive from fetal liver (FL) HSCs and while both populations perform multilineage hematopoiesis they differ in key aspects such as cell cycle status, self-renewal capacity, and transcription factor expression, raising the question how HSCs at earlier developmental stages handle calcium. We employed single-cell perfusion live imaging of calcium dyes and flow cytometry of genetically-encoded calcium indicators to show that FL HSCs possess approximately two-fold increased intracellular calcium from ABM HSCs. Perfusion live imaging also showed increased store-operated calcium entry (SOCE) and decreased calcium efflux through Plasma Membrane Calcium ATPases (PMCAs) from fetal to adult stages. Consistent with these changes, FL HSCs displayed decreased expression of the PMCA gene Atp2b4 and decreased pan-PMCA protein expression. FL HSCs also showed increased nuclear localization of the transcription factor NFAT1, which translocates to the nucleus upon dephosphorylation by the calcium-dependent phosphatase Calcineurin, consistent with increased calcium of FL HSCs. Finally, we show that NFAT1 localization is controlled in part by cytosolic calcium levels, and only ABM HSCs respond to low extracellular calcium to decrease nuclear NFAT localization. These data indicate profound, developmentally regulated changes in calcium physiology between FL and ABM HSCs, which may potentially explain other functional differences between these populations.