The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34 + lin hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well can receive a single hematopoietic stem cell candidate. Sequential movement of each cell-containing well to a microscopic imaging system, serially over a several-day to several-week experiment, is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Bio-box built on the microscope stage. New image analysis software facilitates in the tracking of cell movement, recording of the time of cell division, and immunophenotyping of each of multiple individual or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and also may be applicable to a wide range of other systems of interest in tissue engineering.