Abstract: To maintain self‐renewal and multilineage differentiation capacity, hematopoietic stem cell (HSC) proliferation requires both symmetric and asymmetric cell divisions. We have applied a time‐lapse camera system and our single‐cell culture to correlate early replication behavior with short‐ and long‐term function. Using five‐dimensional flow cytometry to purify subpopulations of fetal liver (FLV), fetal bone marrow (FBM), umbilical cord blood (UCB), adult bone marrow (ABM), and mobilized peripheral blood (MPB), we studied the relationship between colony efficiency (CE) growth pattern and ontogenic age. The highest CE was found among HSC candidates from FLV, FBM, and UCB and the lowest from ABM. Relating the divisional behavior with functional readouts, we demonstrated that although mitotic rate, colony efficiency, and percent of asymmetric divisions all decreased with ontogenic age, the fraction of cells undergoing asymmetric divisions was consistently at 45%. After 10 days of culture, 60.6 ± 9.8% of the PKH bright cells gave rise to colonies (15.8 ± 7.8% dispersed) compared to 15.9 ± 11.1% of the PKH dim cells (2.5 ± 2.5% dispersed). In addition, the much more primitive Myeloid‐Lymphoid Initiating Cells (ML‐IC) are predominantly found in the PKH‐bright population. Thus, primitive function of individual candidate HSCs closely related to their divisional behavior.