We examined the decline in hematopoietic potential observed when human CD34+ cells are cultured in vitro by evaluating the association between proliferation history and the fate of long-term hematopoietic culture-initiating cells (LTHC-ICs) as well as the onset of programmed cell death. The membrane dye PKH2 was used to track ex vivo expanded human CD34+ cells from bone marrow, cord blood, and mobilized peripheral blood, and to identify and isolate CD34+ cells that had divided once, twice, three, or four times or more, as well as cells that had remained cytokine nonresponsive and therefore failed to proliferate. These isolated groups of cells were assayed for their hematopoietic potential, cell cycle status, and percentage of apoptotic cells. A gradual decline in the content of LTHC-ICs, as well as in their ability to initiate and sustain in vitro hematopoiesis, was found to correlate with the number of in vitro cellular divisions, such that the hematopoietic potential of CD34+ cells dividing four or more times was nearly depleted. DNA analysis revealed that cells dividing more than three times resided predominantly in G0/G1 phases of the cell cycle. In addition, the percentage of CD34+ cells undergoing apoptosis was found to increase concomitantly with the number of in vitro cellular divisions; less than 10% of cells dividing once were apoptotic, whereas more than 25% of CD34+ cells dividing four or more times underwent programmed cell death. Together, these data suggest that a proliferation-associated, and possibly activation-induced, loss of hematopoietic potential among dividing CD34+ cells may result from an increase in programmed cell death among dividing primitive hematopoietic progenitor cells.