Abstract SHIP plays an important role in the maturation and DC-induced Ag-specific T cell proliferation downstream of MyD88-independent signaling pathways in Flt3L-DCs. We demonstrate herein that SHIP negatively regulates the proliferation, differentiation, and survival of FL-DCs from BM precursors, as shown by a more rapid appearance and higher numbers of CD11c+ DCs from SHIP−/− cultures as well as increased survival of mature FL-DCs in the absence of Flt3L. This increased survival, which is lost with low levels of the PI3K inhibitor, LY, correlates with an enhanced constitutive activation of the Akt pathway. Interestingly, however, these SHIP−/− FL-DCs display a less-mature phenotype after TLR ligand stimulation, as far as MHCII, CD40, and CD86 are concerned. Unexpectedly, SHIP−/− FL-DCs activated with TLR ligands, which use MyD88-independent pathways, are markedly impaired in their ability to stimulate Ag-specific T cell proliferation, and SHIP−/− FL-DCs activated by TLRs, which exclusively use the MyD88-dependent pathway, are as capable as WT FL-DCs. There is also a more pronounced TH1 skewing by the SHIP−/− FL-DCs than by WT FL-DCs, which is consistent with our finding that SHIP−/− FL-DCs secrete higher levels of IL-12 and TNF-α in response to LPS or dsRNA than their WT counterparts. These results suggest that SHIP negatively regulates FL-DC generation but positively regulates the maturation and function of FL-DCs induced by TLRs, which operate via MyD88-independent pathways.