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Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca²⁺‐releasing messenger that increases the intracellular Ca²⁺ concentration by mobilizing the lysosomal Ca²⁺ store through two‐pore channels 1 (TPC1) and 2 (TPC2). NAADP‐induced lysosomal Ca²⁺ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca²⁺ pool endowed with TPC1 is also present in human endothelial colony‐forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca²⁺ store and TPC1 in circulating ECFCs by harnessing Ca²⁺ imaging and molecular biology techniques. The lysosomotropic agent, Gly–Phe β‐naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca²⁺ sequestration by acidic organelles, caused endogenous Ca² …