Hematopoietic stem cells (HSCs) transplantation and/or the infusion of hematopoietic cells are currently the best alternative for the treatment of a wide variety of hematological disorders. Novel modulators of HSCs fate continue therefore to be identified in an attempt to optimize the in vitro culture of these cells. In this study, main olive leaf components, oleuropein, apigenin 7-glucoside, and luteolin 7-glucoside, and their combination, were investigated for their effect on the viability, self-renewal and differentiation of CD34+ hematopoietic cells. High concentrations of up to 50 μM of these olive leaf phytochemicals, as well as their combination, did not decrease CD34+ cell viability suggesting that these compounds are not cytotoxic on HSCs. Flow cytometric analysis revealed a decrease in the expression of CD34 on the cell surface of HSCs after 9 days of treatment with these compounds indicating a decrease in the proportion of the stem population. Results of the colony-forming unit assay showed an increase in the number of different colonies following treatment with olive leaf phytochemicals. These findings suggest that olive leaf phytochemicals, used alone or in combination, enhance the differentiation of hematopoietic stem cells rather than stimulating their self-renewal providing the first evidence of the potential differentiation effect of main olive leaf compounds on HSCs.