The genome of the bacterium Burkholderia thailandensis encodes for three complete LuxI/LuxR-type quorum sensing (QS) systems: BtaI1/BtaR1 (QS-1), BtaI2/BtaR2 (QS-2), and BtaI3/BtaR3 (QS-3). The LuxR-type transcriptional regulators BtaR1, BtaR2, and BtaR3 modulate the expression of target genes in association with various N-acyl-L-homoserine lactones (AHLs) as signaling molecules produced by the LuxI-type synthases BtaI1, BtaI2, and BtaI3. We have systematically dissected the complex QS circuitry of B. thailandensis strain E264. Direct quantification of octanoyl-homoserine lactone (C8-HSL), N-3-hydroxy-decanoyl-homoserine lactone (3OHC10-HSL), and N-3-hydroxy-octanoyl-homoserine lactone (3OHC8-HSL), the primary AHLs produced by this bacterium, was performed in the wild-type strain and in QS deletion mutants. This was compared to the expression of btaI1, btaI2, and btaI3 using chromosomal mini-CTX-lux transcriptional reporters. Furthermore, transcription of btaR1, btaR2, and btaR3 was monitored by quantitative reverse-transcription PCR (qRT-PCR). We observed that C8-HSL, 3OHC10-HSL, and 3OHC8-HSL are differentially produced over time during bacterial growth and correlate with the btaI1, btaI2, and btaI3 genes expression profiles, revealing a sequential activation of the corresponding QS systems. Moreover, transcription of the btaR1, btaR2, and btaR3 genes is modulated by AHLs, showing that their regulation depend on themselves, and on other systems. We conclude that the three QS systems in B. thailandensis are interdependent, suggesting that they cooperate dynamically and function in a concerted manner in modulating the expression of QS target genes through a sequential regulatory network.