We have previously suggested that phosphatidylinositol 3-kinase (PI3K)/p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells on the basis of analysis of transferrin receptor (Trf-R)-positive (Trf-R(+)) and -negative (Trf-R(-)) cells that appear after treatment with dimethyl sulfoxide (DMSO). In the present study, we analyzed the downstream events of p70 S6K in differentiation and proliferation of both cell types, with a particular focus on c-Myc. Similar to p70 S6K, we found that the expression of c-Myc in Trf-R(+) cells is also higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity, c-Myc expression, and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited p70 S6K activity, c-Myc expression, and G-CSF-dependent proliferation, indicating that the extent of c-Myc inhibition by these inhibitors correlates with a reduction in proliferation, and that c-Myc is downstream from PI3K/p70 S6K. We also determined phosphorylation of the 4E-binding protein 1 (4E-BP1), which is regulated downstream of the mammalian target of rapamycin. The addition of G-CSF failed to enhance the phosphorylation state of 4E-BP1 of HL-60 cells 2 days after DMSO differentiation. An antisense oligonucleotide for c-myc inhibited both G-CSF-dependent enhancement of c-Myc expression and proliferation in Trf-R(+) cells, but did not enhance the differentiation in terms of O(2)(-)-generating ability or fMLP-R expression. In contrast, antisense oligonucleotide for c-myc promoted fMLP-R on non-treated HL-60 cells. We therefore conclude that the PI3K/p70 S6K/c-Myc cascade plays an important role in neutrophilic proliferation in HL-60 cells. Unlike that of rapamycin, however, the antisense oligonucleotide for c-myc could not promote differentiation of Trf-R(+) cells cultured with G-CSF, indicating that another target downstream of p70 S6K may control the differentiation of HL-60 cells in terms of the signal transduction of G-CSF.