Increasing evidences have demonstrated that the invertebrate gill is a predominant tissue participating in the immune response during pathogen challenge. In the present study, the hematopoiesis and immune activities in gill of Pacific oyster Crassostrea gigas were investigated. Stem-like cells with big nuclei and thin cytoplasm were found in the tubules of gill filaments, where DNA synthesis is active and hemocytes production are exuberant. The oysters primarily stimulated by formaldehyde-killed Vibrio splendidus exhibited stronger immune responses and enhanced cell regeneration in gill when they encountered the secondary challenge of live V. splendidus. After the secondary stimulation with V. splendidus, the expression levels of CgClec-4 and CgIFN in the gill of oysters pre-stimulated with formaldehyde-killed V. splendidus were significantly higher (p < 0.05) than that in the oysters pre-stimulated with filter-sterilized (0.22 μm pore size) sea water, while the expression level of CgIL-17 was significantly decreased (p < 0.05). Meanwhile, the protein expression level of hematopoietic transcription factor CgGATA3 and immune-related protein CgEcSOD in gill increased apparently after the secondary challenge with V. splendidus. ROS production was also enhanced (p < 0.05) at 6 h and 24 h after the secondary challenge. The phagocytic rate in gill of oysters pre-stimulated with formaldehyde-killed V. splendidus was significantly increased (p < 0.05) at 6 h after the secondary challenge with live V. splendidus, showing faster response than that pre-stimulated with filter-sterilized sea water. These results collectively showed that the immune parameters in gill were apparently enhanced after secondary challenge with live V. splendidus, indicating that hematopoiesis might participate in immune priming in Pacific oyster C. gigas.