Spliceosome activation is the process of creating the catalytic site for RNA splicing and occurs de novo on each intron following spliceosome assembly. Dozens of factors bind to or are released from the activating spliceosome including the Lsm2-8 heteroheptameric ring that binds the U6 small nuclear RNA 3′-end. Lsm2-8 must be released to permit active site stabilization by the Prp19-containing complex (NineTeen Complex, NTC); however, little is known about the temporal order of events and dynamic interactions that lead up to and follow Lsm2-8 release. We have used colocalization single molecule spectroscopy (CoSMoS) to visualize Lsm2-8 dynamics during activation of Saccharomyces cerevisiae spliceosomes in vitro. Lsm2-8 is recruited as a component of the tri-snRNP and is released after integration of the Prp19-containing complex (NTC). Despite Lsm2-8 and the NTC being mutually exclusive in existing cryo-EM structures of yeast B complex spliceosomes, we identify a transient intermediate containing both ( B Lsm + NTC Δ U4 ) and provide a kinetic framework for its formation and transformation during activation. Prior to B Lsm + NTC Δ U4 assembly, the NTC rapidly and reversibly samples the spliceosome suggesting a mechanism for preventing NTC sequestration by defective spliceosomes that fail to properly activate. In complementary ensemble assays, we show that a base-pairing-dependent ternary complex can form between Lsm2-8 and U2 and U6 helix II RNAs. We propose that this interaction may play a role in formation of transient spliceosome intermediates formed during activation.