Spontaneous maturation observed in dendritic cell (DC) cultures has been linked to their capacity to induce immune responses. Despite several recent studies, the mechanisms and signals triggering spontaneous maturation of DCs are largely unknown. We found that the absence of SWAP-70 causes spontaneous maturation of spleen- and bone marrow-derived DCs and, in vivo, of spleen-resident CD11c(+)CD11b(+)CD8α(-) DCs. Activation markers, cross-presentation of exogenous Ags, and activation of CD8(+) T cells are much increased in Swap-70(-/-) DCs. Spontaneous maturation of Swap-70(-/-) DCs depends on cell-cell contact and does not involve β-catenin signaling. SWAP-70 is known to regulate integrin activity. Signaling through the integrin CD11b (αM) subunit increases spontaneous maturation of wild-type (wt), but not of Swap-70(-/-) DCs. Signaling through the CD18 (β2) subunit decreases spontaneous maturation of wt and Swap-70(-/-) DCs. Constitutive activation of RhoA in Swap-70(-/-) DCs was determined as a key mechanism causing the increased spontaneous maturation. Inhibition of RhoA early, but not late, in the activation process reduces spontaneous maturation in Swap-70(-/-) DCs to wt levels. Inhibition of RhoA activation during CD11b integrin activation had a significant effect only in Swap-70(-/-) but not in wt DCs. Together, our data suggest that integrin-mediated spontaneous maturation of wt DCs does not depend on active RhoA, whereas the increase in spontaneous maturation of Swap-70(-/-) DCs is supported by integrin CD11b and by hyperactive RhoA. Thus, SWAP-70 deficiency reveals two pathways that contribute to spontaneous maturation of DCs.