Granulocyte colony-stimulating factor (G-CSF) is widely used in clinical settings to mobilize hematopoietic stem cells (HSCs) into the circulation for HSC harvesting and transplantation. However, whether G-CSF directly stimulates HSCs to change their cell cycle state and fate is controversial. HSCs are a heterogeneous population consisting of different types of HSCs, such as myeloid-biased HSCs and lymphoid-biased HSCs. We hypothesized that G-CSF has different effects on different types of HSCs. To verify this, we performed serum-free single-cell culture and competitive repopulation with cultured cells. Single highly purified HSCs and hematopoietic progenitor cells (HPCs) were cultured with stem cell factor (SCF), SCF + G-CSF, SCF + granulocyte/macrophage (GM)-CSF, or SCF + thrombopoietin (TPO) for 7 days. Compared with SCF alone, SCF + G-CSF increased the number of divisions of cells from the lymphoid-biased HSC-enriched population but not that of cells from the My-bi HSC-enriched population. SCF + G-CSF enhanced the level of reconstitution of lymphoid-biased HSCs but not that of myeloid-biased HSCs. Clonal transplantation assay also showed that SCF + G-CSF did not increase the frequency of myeloid-biased HSCs. These data showed that G-CSF directly acted on lymphoid-biased HSCs but not myeloid-biased HSCs. Our study also revised the cytokine network at early stages of hematopoiesis: SCF directly acted on myeloid-biased HSCs; TPO directly acted on myeloid-biased HSCs and lymphoid-biased HSCs; and GM-CSF acted only on HPCs. Early hematopoiesis is controlled differentially and sequentially by a number of cytokines.