Various combinations of cytokines have profoundly different effects on inhibition of apoptosis and stimulation of self-renewal division of hematopoietic stem cells (HSC) in short-term, ex vivo culture. Our goal was to quantitate expansion of cells with a primitive CD34+ Thy-1+ phenotype, as well as cell cycling, division history, differentiation, and apoptosis of CD34+ cells enriched from normal donor mobilized peripheral blood (MPB) cells. The balance of these parameters determines the net number of transplantable HSC produced in ex vivo cultures. Comparing several different combinations of cytokines added to 90-hour cultures of MPB CD34 cells, thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL) gave the best result, with the lowest percentage of apoptotic cells and a mean 1.2-fold increase in the number of CD34+ Thy-1+ cells. A combination of interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) gave the worst outcome, including a decrease of CD34+ Thy-1+ cell number to a mean of 30% of the starting cell number. Cell division history was tracked using the dye 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE). Division of CD34+ Thy-1+ cells was faster and more synchronous in TPO, FL, and KL than in IL-3, IL-6, and LIF, which left a significant proportion of CD34+ cells undivided. Such detailed analyses of short-term, ex vivo cultures generated "replication scores," which allowed prediction of a sixfold improvement of the efficiency of gene transduction of primitive hematopoietic progenitors from MPB, using TPO, FL, and KL to replace IL-3, IL-6, and LIF. Analysis of retroviral transduction efficiency confirmed the increase of transgene expression from MPB primitive hematopoietic progenitors assayed after stromal culture was fivefold, validating the usefulness of multiparameter analysis of short-term cultures for survival and replication of CD34+ Thy-1+ cells.