The transplantation of hematopoietic stem and progenitor cells (HSPC) is an established lifesaving therapy. Bone marrow (BM), harvested from heparinized cadaveric organ donors, peripheral blood (PB) and cord blood (CB), are important sources of hematopoietic stem cells. HSPCs, which are used for transplantation purposes, are routinely evaluated in terms of number of mononuclear cells (MNCs), CD34+ MNCs count and viability. The efficacy of grafting is determined additionally in clonogenic tests in vitro. These tests deliver important information about the number of HSPCs and their proliferative potential. Unfortunately, they do not give a possibility to evaluate the functional HSPC chemotactic reactivity in the SDF-1 gradient, which is probably the key phenomenon for HSPC homing after transplantation procedure. Thus, the aim of our study was to optimize HSPC isolation according to their chemotactic reactivity in SDF-1 gradient. Using multiparameter cell sorter (FACS Aria, BD) we examined the HSPCs attracted by SDF-1 on a single cell level. The population of cells which participated in the chemotactic process was highly enriched in CXCR4+lin-AC133+CD45+ cells (referred as hematopoietic stem cells) and to our surprise in CXCR4+lin-AC133+CD45- cells (referred as pluripotent stem cells) in quantitative amounts. Since reactivity of HSPCs may depend on various factors involved in the protocol of their isolation and short-term storage, we tested the most commonly used anticoagulants (ACD, CPDA-1, EDTA and Heparin) and culture media (DME, IMDM, RPMI). HSPCs, harvested from CB, PB and BM, were subsequently investigated for clonogenic growth of CFU-GM in methylcellulose cultures and for the level of apoptosis by employing annexin V staining. Evaluating clonogenic potential, ability of chemotactic reactivity in SDF-1 gradient and intensification of apoptosis of HSPC as the most safe anticoagulant and medium were selected. This study has proved that chemotactic reactivity of HSPCs is a new but very important parameter which should be included in the procedure of their isolation.