The effects of platelet factor 4 (PF4) on the viability and chemosensitivity of normal hematopoietic cells and cancer cell lines were studied to determine the mechanisms whereby PF4 functions as either an inhibitor or a protector and to evaluate its clinical significance. Two other chemokines, interleukin-8 (IL-8) and neutrophil-activating peptide-2 (NAP-2), were also studied in comparison to PF4. Using a tetrazolium salt assay for cell viability, we observed that PF4 at 1 to 50 μg/mL supported the viability of normal human bone marrow cells. Approximately 45% of cells cultured for 48 hours survived, whereas 80% or more survived in the presence of PF4 5 μg/mL. PF4 also supported the viability of CD34⁺ cord blood (CB) cells and protected them from apoptosis induced by transforming growth factor β1 (TGFβ1) and cytotoxic drugs. Pretreatment of CD34⁺ cells by PF4, but not by TGFβ1, caused an increase in the number of megakaryocyte colonies after these cells were replated in secondary cultures. Flow cytometry analysis showed that when CD34⁺ cells were preincubated with PF4 or TGFβ1 for 12 days in hematopoietic growth factor–rich medium, an increased number of remaining CD34⁺ cells was observed only for PF4-treated cells. Furthermore, PF4 significantly reduced the chemosensitivity of bone marrow cells, as shown by its ability to increase the 50% inhibition concentration (IC₅₀) of several cytotoxic agents. Like PF4, IL-8 and NAP-2 at 0.1, 0.6, and 1 μg/mL supported the survival of myeloid progenitors, including colony-forming units granulocyte, erythroblast, monocyte, megakaryocyte (CFU-GEMM), CFU-megakaryocyte (CFU-MK), CFU–granulocyte/macrophage (CFU-GM), and burst-forming units–erythroblast (BFU-E), and reduced their sensitivity to the toxicity of etoposide (ETP). Protamine sulfate at 1 to 100 μg/mL showed no such activity of PF4. Interestingly, the three chemokines failed to affect significantly the viability and chemosensitivity of three leukemic and two other tumor cell lines. Based on these results, we conclude for the first time that PF4 and IL-8 and NAP-2 support the survival of normal hematopoietic precursors and protect them from the toxicity of chemotherapeutic agents. Because such activities are unique to normal hematopoietic cells but not to the cancer cell lines evaluated, a potential clinical application of these molecules in the treatment of cancer is suggested.